Alpha-Synuclein Kinase

ABSTRACT

The invention provides agents and methods for treatment of diseases associated with Lewy body diseases (LBDs) in the brain of a patient. Preferred agents include inhibitors of PLK2 kinase.

RELATED APPLICATIONS

This application claims priority as a continuation-in-part of U.S.patent application Ser. No. 11/669,093, filed Jan. 30, 2007, whichclaims benefit of U.S. provisional application No. 60/764,000, filedJan. 31, 2006, each of which is herein incorporated by reference in itsentirety for all purposes.

BACKGROUND OF THE INVENTION

Lewy body diseases (LBDs) are characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al, Clinical andpathological diagnosis of dementia with Lewy bodies (DLB): Report of theCDLB International Workshop, Neurology (1996) 47:1113-24). LBDs includeParkinson's disease, Diffuse Lewy body disease (DLBD), Lewy body variantof Alzheimer's disease (LBV), combined Parkinson's disease (PD) andAlzheimer's disease (AD), and the syndromes identified as multiplesystem atrophy (MSA). Dementia with Lewy bodies (DLB) is a term coinedto reconcile differences in the terminology of LBDs. Disorders with LBscontinue to be a common cause for movement disorders and cognitivedeterioration in the aging population (Galasko et al.,Clinical-neuropathological correlations in Alzheimer's disease andrelated dementias. Arch. Neurol. (1994) 51:888-95). Although theirincidence continues to increase, creating a serious public healthproblem, to date these disorders lack approved treatments (Tanner etal., Epidemiology of Parkinson's disease and akinetic syndromes, Curr.Opin. Neurol. (2000) 13:427-30). The cause for LBDs is controversial andmultiple factors have been proposed to play a role, including variousneurotoxins and genetic susceptibility factors.

In recent years, new hope for understanding the pathogenesis of LBDs hasemerged. Specifically, several studies have shown that the synapticprotein alpha-synuclein plays a central role in PD pathogenesis because:(1) this protein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi etal., Neurosci. Lett. (1997) 239:45-8), (2) mutations in thealpha-synuclein gene co-segregate with rare familial forms ofparkinsonism (Kruger et al., Nature Gen. (1998) 18:106-8;Polymeropoulos, et al., Science (1997) 276:2045-7) and, (3)overexpression of alpha-synuclein in transgenic mice (Masliah et al.,Science (2000) 287:1265-9) and Drosophila (Feany et al, Nature (2000)404:394-8) mimics several pathological aspects of PD.

Many scientists believe that PD is a relatively late development in asystemic synucleinopathy and that “parkinsonism is just the tip of theiceberg” (Langston, Annals of Neurology (2006) 59:591-596). For example,Lewy bodies have been described in sympathetic ganglia and in themyenteric plexus of the gut (Herzog E., Dtch Z Nervenheilk (1928)107:75-80; Kupsky et al., Neurology (1987) 37:1253-1255). Variousdisorders have been associated with the presence of Lewy bodies. Forexample, Lewy bodies have been found in the brain stem of a patient withrapid eye movement sleep behavioral disorder (Uchiyama et al., Neurology(1995) 45:709-712). Olfactory dysfunction has been reported in many PDpatients long before the development of parkinsonism. Examination ofcardiac tissue from patients with incidental Lewy body disease andtypical PD revealed synuclein-positive neuritis in the myocardium(Iwanaga et al., Neurology (1999) 52:1269-1271). There is also evidencethat esophageal, lower bowel and bladder dysfunction are earlymanifestations of PD-related pathology in the peripheral autonomicsystem (Qualman et al., Gastroenterology (1984) 87:848-856; Castell etal., Neurogasdtroenterol Motil (2001) 13:361-364; Hague et al., ActaNeuropathol (Berl) (1997) 94:192-196). Thus, the fact that accumulationof alpha-synuclein in the brain and other tissues is associated withsimilar morphological and neurological alterations in species as diverseas humans, mice, and flies suggests that this molecule contributes tothe development of PD.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of screening an agent foractivity for treating a Lewy Body disease (LBD). Such diseases includeParkinson's disease (PD), Diffuse Lewy body disease (DLBD), Lewy bodyvariant of Alzheimer's disease (LBV), combined PD and Alzheimer'sdisease (AD), and the syndromes identified as multiple system atrophy(MSA). Some methods entail identifying an agent that modulates theactivity or expression of a kinase shown in Table 1A, B; C, Table 2,Table 11 or Table 12, and determining whether the agent shows activityuseful in treating LBD in an animal model of the disease. In somemethods the modulation is inhibition. In some methods, step (a) involvesidentifying whether the agent inhibits the kinase. In some methods, step(a) is performed in a cell transformed with a nucleic acid expressingthe kinase and/or alpha-synuclein. In some methods, step (a) isperformed in vitro. In some methods, step (b) is performed in atransgenic animal model of LBD disease, and the transgenic animal mayhave a transgene expressing human alpha-synuclein. Preferably, thekinase is at least one of: APEG1, PLK2, CDC7L1, PRKG1, MAPK13, GAK,RHOK, ADRBK1, ADRBK2, GRK2L, GRK5, GRK6, GRK7, IKBKB, CKII and MET andthe modulation is inhibition. More preferably, the kinase is PLK2 orGRK6 and the modulation is inhibition. More preferably, the kinase isPLK2. Preferably in some methods, the kinase is PRKG1, MAPK13, or GAKand the modulation is activation. In some aspects, step (b) involvescontacting the transgenic animal with the agent and determining whetherthe agent inhibits formation of deposits of alpha-synuclein relative toa control transgenic animal not treated with the agent.

In another aspect, the invention provides methods of effecting treatmentor prophylaxis of a LBD. Some examples of the method involveadministering to a patient suffering from or at risk of the disease, aneffective regime of an agent effective to modulate activity orexpression of a kinase. The kinase can be one of those shown in Table1A, B or C, Table 2, Table 11 or Table 12. Preferably, the agent is anantibody to the kinase, a zinc finger protein that modulates expressionof the kinase, or an antisense RNA, siRNA, ribozyme or RNA having asequence complementary to a nucleic acid sequence of the kinase. In somemethods, the modulation is inhibition, and preferably, the kinase is atleast one of the following: APEG1, PLK2, CDC7L1, RHOK, ADRBK1, ADRBK2,GRK2L, GRK5, GRK6, GRK7, IKBKB, CKII and MET. More preferably, thekinase is PLK2 or GRK6. More preferably, the kinase is PLK2. In some ofthe methods, the kinase is at least one of: PRKG1, MAPK13, and GAK, andthe modulation is activation.

In one aspect the invention provides method of treating a patientdiagnosed with Parkinson's Disease by administering a therapeuticallyeffective amount of an agent that inhibits PLK2 activity. In anembodiment the agent preferentially inhibits PLK2 activity relative toinhibition of PLK1 activity and/or PLK3 activity and/or PLK4 activity.The agent may be, for example, an siRNA. In one embodiment the patientis not diagnosed or under treatment for cancer and/or is not diagnosedor under treatment for Alzheimer's disease.

In related aspects, the invention provides a method for inhibitingphosphorylation of alpha-synuclein in a mammalian cell by reducingpolo-like kinase 2 (PLK2) activity in the cell such that phosphorylationof synuclein is reduced. In a related aspect, the invention provides amethod for inhibiting phosphorylation of alpha-synuclein in a mammaliancell (e.g., a neuronal cell) by contacting the cell with a compound thatreduces PLK2 activity in the cell such that phosphorylation ofalpha-synuclein is reduced. For example, the agent may reduce expressionof a PLK2 gene product.

In certain embodiments the agent preferentially reduces PLK2 activityrelative to reduction of PLK1 activity, PLK2 activity, or PLK3 activity.In certain embodiments the agent has a molecular weight less than 4000.In one embodiment the agent is a synthetic compound. In some embodimentsthe agent is a polynucleotide that inhibits expression or translation ofa PLK2 RNA transcript, such as an siRNA. In an embodiment, one strand ofthe double stranded region of the siRNA is perfectly complementary to aPLK2 transcript but not to a PLK1 transcript or a PLK3 transcript.

In another aspect, the invention provides methods of identifying akinase that phosphorylates alpha-synuclein by transfecting a cellexpressing alpha-synuclein with a nucleic acid having a sequencecomplementary to a gene encoding a kinase or zinc finger protein thatspecifically binds to the gene. The transfected nucleic acid or zincfinger protein inhibits expression of the kinase; and an amount ofphosphorylated alpha-synuclein the cell can then be measured relative toa control cell not transfected with the siRNA or nucleic acid encodingthe same. In this case, a reduction in phosphorylated alpha-synucleinwill provide an indication that the kinase phosphorylatesalpha-synuclein. Some methods also include measuring an amount ofalpha-synuclein produced by the cell relative to a control cell nottransfected with the nucleic acid. In some methods, the nucleic acid isan siRNA or a DNA molecule encoding the same.

The invention provides a method of identifying an agent reducesalpha-synuclein phosphorylation in a mammalian cell expressingalpha-synuclein. The method includes selecting an agent that a) reducesactivity of PLK2 in a cell expressing PLK2 (and optionally expressingsynuclein), and b) does not reduce activity of PLK1 in a cell expressingPLK1, or reduces activity of PLK1 at a higher EC₅₀ than for PLK2; and/orc) does not reduce activity of PLK3 in a cell expressing PLK3, orreduces activity of PLK3 at a higher EC₅₀ than for PLK2; and/or d) doesnot reduce activity of PLK4 in a cell expressing PLK4, or reducesactivity of PLK4 at a higher EC₅₀ than for PLK2. The cell can be amammalian cell overexpressing alpha-synuclein. In one embodiment theagent a) reduces activity of PLK2 in a cell expressing PLK2; b) does notreduce activity of PLK1 in a cell expressing PLK1, or reduces activityof PLK1 at a higher EC₅₀ than for PLK2; c) does not reduce activity ofPLK3 in a cell expressing PLK3, or reduces activity of PLK3 at a higherEC₅₀ than for PLK2; and d) does not reduce activity of PLK4 in a cellexpressing PLK4, or reduces activity of PLK4 at a higher EC₅₀ than forPLK2. In a further step, the method involves determining whether theselected agent shows activity useful in treating Lewy Body Disease in ananimal model of the disease or a cellular model of the disease. Animalmodels include transgenic animals. Cellular models includeneuronally-derived cell cultures and mammalian cells over-expressingalpha-synuclein. Activities that can be assayed include reduction of theproportion of total alpha-synuclein that is phosphorylated at serine-129or a reduction in aggregation of alpha-synuclein in the cell.

In other aspects, the invention provides methods of method of screeningan agent for activity for treating a Lewy Body disease (LBD), byidentifying an agent that modulates the activity or expression ofsynphilin, and determining whether the agent shows activity useful intreating LBD in an animal model of the disease.

In other aspects, the invention provides methods for producing Ser-129phosphorylated-alpha synuclein, by providing a plasmid encodingalpha-synuclein and a plasmid encoding PLK2 in a bacterial cell,culturing the cell so that the plasmids are co-expressing to producealpha synuclein and PLK2 so that the PLK2 phosphorylates thealpha-synuclein in a bacterial cell, and isolating phosphorylatedalpha-synuclein from the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the results of the in vitro phosphorylation assay foralpha-synuclein phosphorylation by a variety of recombinant kinases.FIG. 1A shows total alpha-synuclein, FIG. 1B shows phosphorylation ofthe pser-129 (phospho-serine-129) of alpha-synuclein and FIG. 1C showsphosphorylation of the pser-87 (phospho-serine-87) of alpha-synuclein.

FIGS. 1D-F show a study with recombinant kinases, including kinases fromthe GPCR-receptor kinase (GRK) family and PLK2. FIG. 1D shows totalalpha-synuclein, FIG. 1E shows phosphorylation of the pser-129 ofalpha-synuclein and FIG. 1F shows phosphorylation of the pser-87 ofalpha-synuclein.

FIGS. 2A and B show the results of kinase activity in vitro for variouskinases.

FIG. 2A shows the total (AS). FIG. 2B shows phosphor-serine 129.

FIGS. 3A-C show the results of kinase activity in vitro for variouskinases. FIG. 2A shows the total AS. FIG. 3B shows Serine 129. FIG. 3Cshows phospho-serine 87.

FIGS. 4A and B show the effect of phospholipid on the assay results inFIGS. 3A and 3B. FIG. 4A shows the total AS. FIG. 4B shows Serine 129.

FIG. 5 shows the results of transfection of cDNA to PLK2 into293-synuclein cells. Cells were analyzed by ELISA for total andphospho-synuclein levels.

FIG. 6 shows the results of transfection of cDNA to GPRK6 and PLK2 intoHEK-Synuclein cells.

FIG. 7 shows that knockdown of the PLK2 using siRNA from a second sourcecauses a reduction in the proportion of alph-synuclein that isphosphorylated.

FIGS. 8A and 8B show the in vitro phosphorylation of alpha-synuclein byputative kinase targets in alpha-synuclein KO mouse brain.

FIGS. 9A and 9B show the in vitro phosphorylation of alpha-synuclein byputative kinase targets in alpha-synuclein KO mouse brain.

FIG. 10 shows that siRNA knockdown of PLK2, but not PLK3 or PLK4,reduced alpha-synuclein phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “agent” is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity.

A “pharmacological” activity means that an agent exhibits an activity ina screening system that indicates that the agent is or may be useful inthe prophylaxis or treatment of a disease. The screening system can bein vitro, cellular, animal or human. Agents can be described as havingpharmacological activity notwithstanding that further testing may berequired to establish actual prophylactic or therapeutic utility intreatment of a disease.

A Lewy-like body is a deposit of alpha-synuclein found in a transgenicanimal that resembles some or all of the characteristics of a Lewy bodyfound in human patients. The preferred characteristics are a compactalpha-synuclein positive inclusion. These inclusions preferably form inan age-dependent manner. The formation of alpha-synuclein positiveinclusions preferably results in observable cellular pathology, leadingto loss of functionality of affected neurons. Loss of function ofaffected neurons can be determined through behavioral tests,neuropharmacological response evaluation and electrophysiology.

The phrase “specifically binds” refers to a binding reaction which isdeterminative of the presence of the protein in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated conditions, a specified ligand binds preferentially to aparticular protein and does not bind in a significant amount to otherproteins present in the sample. A molecule such as an antibody thatspecifically binds to a protein often has an association constant of atleast 10⁶ M⁻¹ or 10⁷ M⁻¹, preferably 10⁸ M⁻¹ to 10⁹ M⁻¹, and morepreferably, about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See, e.g., Harlow and Lane(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, for examples of immunoassay formats and conditions that can beused to determine specific immunoreactivity.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequent coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra.). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. For identifying whether a nucleic acid or polypeptide is withinthe scope of the invention, the default parameters of the BLAST programsare suitable. The BLASTN program (for nucleotide sequences) uses asdefaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a word length (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix. The TBLATN program (using proteinsequence for nucleotide sequence) uses as defaults a word length (W) of3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

For purposes of classifying amino acids substitutions as conservative ornon-conservative, amino acids are grouped as follows: Group I(hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gln, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at leastabout 90%, at least about 95%, or at least about 99% w/w purity.However, using conventional protein purification techniques, homogeneouspeptides of at least 99% w/w can be obtained.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragment including separate heavy chains, lightchains Fab, Fab′F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992).

A symptom of a disorder means a phenomenon experienced by an individualhaving the disorder indicating a departure from normal function,sensation or appearance.

A sign of a disorder is any bodily manifestation that serves to indicatepresence or risk of a disorder.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, “treating” a condition (e.g., Parkinson's Disease) orpatient refers to taking steps to obtain beneficial or desired result.For purposes of this invention, beneficial or desired results include,but are not limited to, alleviation or amelioration of one or moresymptoms of Parkinson's Disease, diminishment of extent of disease,delay or slowing of disease progression, amelioration, palliation orstabilization of the disease state.

As used herein, a “therapeutically effective amount” of a drug is anamount of a drug that, when administered to a subject diagnosed withParkinson's disease, or diagnosed as being at high risk for developingParkinson's disease will have the intended therapeutic effect, e.g.,alleviation, amelioration, palliation or elimination of one or moremanifestations of the disease in the subject. The full therapeuticeffect does not necessarily occur by administration of one dose, and mayoccur only after administration of a series of doses. Thus, atherapeutically effective amount may be administered in one or moreadministrations.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises alpha-synuclein peptide encompasses both anisolated alpha-synuclein peptide and alpha-synuclein peptide as acomponent of a larger polypeptide sequence.

Unless otherwise apparent from the context, each embodiment, element,step or feature of the invention can be used in combination with anyother.

II. General

The invention is premised in part on the insight that Lewy Body diseases(LBDs) can be inhibited by inhibiting one or more kinases thatphosphorylate alpha-synuclein and/or inhibit its production. Althoughpractice of the invention is not dependent on an understanding ofmechanism, it is believed that phosphorylation of alpha-synuclein atserine-129 is one of a series of molecular events leading to formationof intracellular deposits of alpha-synuclein. Alpha-synucleinphosphorylated at ser-129 is highly enriched in Lewy bodies (LBs) inDiffuse Lewy body disease (DLBD), multiple system atrophy (MSA) andfamilial forms of Parkinson's Disease (PD). The abnormal accumulation ofphospho-alpha-synuclein in LBs indicates that phospho-synuclein may be apathogenic species that drives LB formation, and that the kinase(s)responsible for its phosphorylation or which regulates production ofalpha-synuclein itself are therapeutic target(s) for treatment ofmultiple synucleinopathies. Other events in this series likely includeproteolytic cleavages following phosphorylation (see WO 2005/013889,filed May 19, 2004).

Identification of the kinase(s) primarily responsible forphosphorylation of alpha-synuclein allows compounds that reduce activityof the relevant kinase(s) to be identified. For convenience, referenceherein to “phosphorylation of alpha-synuclein” refers to phosphorylationat Serine-129 (but does not exclude additional phosphorylationelsewhere, e.g., Serine-87).

The present application reports identification of several kinases wherereduction in kinase activity is accompanied by a reduction ofphosphorylation of alpha-synuclein and/or a reduction in totalalpha-synuclein level. In particular, the kinase PLK2 can be inhibitedto reduce phosphorylation of alpha-synuclein. The invention providesmethods of (i) identifying modulators of the activity and expression ofthese kinases, (ii) methods of treating Lewy body diseases using kinaseinhibitors, and (iii) exemplary kinase inhibitors for use in treatingLewy body diseases.

As discussed in the Examples, infra, we have carried out a variety ofexperiments to identify kinases important in phosphorylation ofalpha-synuclein. Section III outlines a strategy for identifyingalpha-synuclein kinases. Section IV summarizes results of screeningassays used to identify likely alpha-synuclein kinases. Section Vdescribes agents that reduce synuclein kinase activity or expression andmay be used therapeutically. Section VI describes methods for treatingParkinson's Disease and other Lewy Body Diseases. Section VII describesLewy Body Diseases. Section VIII describes transgenic animal andcellular models of Lewy Body Disease. Section IX describes method foridentification of modulators of PLK2 and other kinases. Section Xdescribes methods for alpha-synuclein isolation. Section XI providesexperimental results including the aforementioned screening assays.

III. Identification of Target Kinases

Kinases that directly or indirectly modulate phosphorylation ofalpha-synuclein can be identified as shown in the Examples. In general,a library of potential inhibitors is designed based on the knownsequences of a collection of kinase genes. The members of the librarycan be any of the types of molecule described above. Members of thelibrary are then introduced into cells expressing alpha-synuclein.Preferably both the cells and the alpha-synuclein are human. Usually,such cells are transfected with both DNA encoding human alpha-synucleinand DNA encoding the library member to be tested. Library members can bescreened individually or en masse. After introduction of a librarymember, and culturing for a period sufficient for the library member tobe expressed and effect repression of its kinase, the levels of totalalpha-synuclein and phosphorylated alpha-synuclein are measured andcompared with corresponding levels in an otherwise similar control cellnot treated with a library member to suppress expression of a kinase.Measurements can be made by immunoassay using an antibody specific foralpha-synuclein (preferably human alpha-synuclein) to measure totallevels of alpha-synuclein, and an antibody specific for phosphorylatedalpha-synuclein to measure the level of phosphorylated alpha-synuclein.Exemplary antibodies are described in WO05047860, incorporated herein byreference. A reduction in level of phosphorylated alpha-synucleinbetween the treated and control cell that is significant in the sense ofbeing outside the typical margin of error for measurements, indicatesthat the inhibitor introduced into the cell inhibited a kinase, whichdirectly or indirectly affected phosphorylation of alpha-synuclein. Theidentity of the kinase can be determined from the identity of inhibitor,either by screening inhibitors individually, or if inhibitors arescreened en masse, by sequencing the nucleic acid encoding theinhibitor. Likewise a reduction in the total level of alpha-synucleinbetween treated and control cells that is outside the margin of typicalexperimental error in measuring such levels provides an indication thatthe inhibitor inhibits a kinase that indirectly affects the expressionlevel of alpha-synuclein.

Kinases identified by the initial screen, particularly, kinases known tobe serine kinases, can then be tested for their capacity tophosphorylate alpha-synuclein in vitro, in cells or in transgenic animalmodels. An in vitro assay is an indication of whether a kinase directlyphosphorylates alpha-synuclein and is therefore only useful for thekinases identified in the initial screen which are thought to be capableof directly phosphorylating alpha-synuclein. Cellular and transgenicassays can be used to screen kinases that affect phosphorylation eitherdirectly or indirectly. In vitro assays may be performed by contactingalpha-synuclein with the kinase under test and ATP in a suitable buffer.Preferably, the ATP is γ-32P ATP, in which case phosphorylatedalpha-synuclein is radiolabeled and can be detected on a gel.Phosphorylation can also be measured using an antibody specific tophosphorylated alpha-synuclein as described before. Alternatively,phosphorylation can be measured indirectly by measuring ATP consumptionusing a coupled assay, in which ADP is detected as described for exampleby Nature 78, 632 (1956); Mol. Pharmacol. 6, 31-40 (1970). The extent ofphosphorylation can be compared with a control in which the kinase orATP or both is/are omitted. An increase in phosphorylation is anindication that the kinase directly phosphorylates alpha-synuclein.Cellular assays are performed on cells expressing alpha-synuclein,preferably human alpha-synuclein transfected into the cells. A nucleicacid capable of expressing the kinase is also transfected into thecells. The level of phosphorylated alpha-synuclein in the cells ismeasured relative to that in similar control cells lacking thetransfected kinase. An increase in phosphorylation is an indication thatthe kinase directly or indirectly phosphorylates alpha-synuclein.Transgenic assays can be performed by comparing a transgenic animalexpressing human alpha-synuclein disposed to develop Lewy body-likedeposits with a similar animal also expressing a kinase transgene. Areduction in phosphorylated alpha-synuclein and/or in Lewy body-likedeposits in the transgenic animal with the additional kinase transgenerelative to the transgenic animal with just the alpha-synucleintransgene is an indication that the kinase is directly or indirectlyinvolved in phosphorylating alpha-synuclein.

IV. Target Kinases

Tables 1A, 1B and 1C show proteins whose inhibition modulates thephosphorylation at position ser-129. Table 1A shows kinases that canphosphorylate serine and/or threonine residues and sometimes tyrosine.Table 1B shows tyrosine kinases that cannot (so far as is known) modifyserine residues. Table 1C shows kinases that phosphorylate non-proteintargets but are not known to phosphorylate proteins. Kinases from theupper portion of Table 1A are candidates for direct phosphorylation ofser-129 of alpha-synuclein. Kinases from the upper part of Table 1B arealso useful therapeutic targets via roles indirectly phosphorylatingalpha-synuclein. Proteins in the upper part of Table 1C are also usefultherapeutic targets for the same reason. Cols. 1, 2 and 3 of each tableindicate the gene name, kinase name and Genbank accession number ofkinases. The next column indicates whether treatment of cells with siRNAto that kinase decreased (“down”) or increased (“up”) phosphorylation ofser-129. The next three columns indicate the number of standarddeviations the measured level of phosphorylation departs from the meanin three independent experiments. The final two columns indicate thekinase family (i.e., amino acid specificity) and group.

Table 2 shows kinases whose inhibition modulates the overall levels ofhuman alpha-synuclein without changing the percentage ofphosphorylation. Table 2 shows all of the kinases with the strongestreduction in levels of human alpha-synuclein. The columns are labeledsimilarly to Tables 1A, 1B and 1C.

Tables 3 and 4 show kinases from Tables 1 and 2 that were confirmed inthe Examples to modulate overall levels of human alpha-synuclein. Thekinases that were verified include PLK2, APEG 1, CDC7L1, MET, GRK1, 2,6, and 7 as kinases that phosphorylate alpha-synuclein directly orindirectly. The kinases that were found to increase alpha-synucleinphosphorylation when inhibited, PRKG1, MAPK13, and GAK, are likely tofunction as negative regulators of alpha-synuclein phosphorylation.Further data from phosphorylation studies in vitro identified PKL2,GRK2, 5, 6, and 7 as capable of phosphorylating alpha-synuclein in vitroand also identified CKII and IKBKB. Further studies in cell cultureshowed that PLK2 and GPRK6 could directly phosphorylate alpha-synucleinin cell culture. These data were substantiated withimmunohistochemistry. In summary, PLK2 and, to a lesser extent, GRK6 areparticularly preferred targets for therapeutic intervention in Lewy bodydiseases because they can directly phosphorylate alpha-synuclein. Agentsthat inhibit PLK2 and GRK6 also inhibit phosphorylation ofalpha-synuclein and thus can be used in treatment or prophylaxis of Lewybody disease.

In the Examples, below, transfection of cells with siRNA and knockdownof specific kinase targets was employed to identify kinases thatmodulated alpha-synuclein phosphorylation directly or indirectly.Subsequent experiments in vitro and in cell culture showed that two ofthese kinases, PLK2 directly and specifically phosphorylated the serine129 of alpha-synuclein. Further experiments showed that PLK2phosphorylated the serine 129 of alpha-synuclein to a much greaterextent than GRK6 and other kinases described herein under theexperimental conditions used. Thus, PLK2 is very likely a synucleinkinase. Additional evidence that PLK2 is a synuclein kinase is providedin Examples 11-16. Synuclein phosphorylation is reduced in cells treatedwith siRNA directed to PLK2, inhibitors of PLK2 activity reducesynuclein phosphorylation in a variety of cell types including primaryneuronal cultures and cells over expressing PLK2, inhibitors affectendogenous kinase in with an EC₅₀ consistent with the EC₅₀ observed fortheir effect on PLK2.

PLK2 is a Polo like kinase that is a G1 cell cycle protein, has a rapidturnover in cells, and is expressed in brain where it is involved insynaptic plasticity. The PLK family members are serine/threoninekinases, and contains four members that have an N-terminal kinase domainand a C-terminal regulatory domain consisting of two (PLKs 1-3) or one(PLK4) polo-box domains. The polo-box domain serves to bind toscaffolding proteins that then target the PLKs to specific sub-cellularlocations and to phosphorylate their target proteins (Seeburg, D. P. etal, Oncogene, 2005). The polo-box also serves to negatively regulate thekinase domain by adopting a conformation that prevents kinase activity.Upon binding of the polo-box to a scaffolding protein, the polo-box isremoved from the kinase domain, whereupon the kinase becomes active andis able to phosphorylate its substrate/s. Polo-like kinases aredescribed in Seeburg et al., 2005, “Polo-like kinases in the nervoussystem” Oncogene 24:292-8; Lowery et al., 2005, “Structure and functionof Polo-like kinases” Oncogene 24:248-59; and Winkles et al., 2005,“Differential regulation of polo-like kinase 1, 2, 3, and 4 geneexpression in mammalian cells and tissues” Oncogene 24:260-6. DNA andprotein sequences can be found at the accession numbers below:

GenBank Accession Entrez UniProt Kinase number Gene ID ID PLK1 NM_0050305347 P53350 PLK2 NM_006622 10769 Q9NYY3 PLK3 AJ293866, NM_004073 1263Q9H4B4 PLK4 Y13115, NM_014264 10733 O00444

When PLK2 is activated, it is targeted to dendrites of activatedneurons, where it is believed to phosphorylate proteins in the synapticterminals. An exemplary accession number for PLK2 is provided Table 1A.The sequence for PLK2 can also be found in any one of Ma, et al. Mol.Cell. Biol. 23 (19), 6936-6943 (2003), Burns, et al. Mol. Cell. Biol. 23(16), 5556-5571 (2003), Matsuda, et al. Oncogene 22 (21), 3307-3318(2003), Shimizu-Yoshida et al. Biochem. Biophys. Res. Commun. 289 (2),491-498 (2001), Liby, et al. DNA seq. 11 (6), 527-533 (2001), Holtrich,et al. Oncogene 19 (42), 4832-4839, Ouyang, et al. Oncogene 18 (44),6029-6036 (1999), and Kauselmann, et al. EMBO J. 18 (20), 5528-5539;reference to an amino acid or nucleic acid sequence of PLK2 includes thesequences of any of these references or allelic variants thereof. PLK2is also called SNK; for consistency, the name PLK2 is used throughoutthe present patent application.

APEG1, CDC7L1, MET, IKBKB, CKII, GRK1, GRK2, GRK6 and GRK7 are alsotargets for therapeutic intervention in Lewy body diseases because theyare likely to be indirect activators of the direct kinase(s). Thus,agents that inhibit APEG1, CDC7L1, MET, IKBKB, CKII, GRK1, GRK2, GRK6and GRK7 also inhibit phosphorylation of alpha-synuclein and can be usedfor treatment or prophylaxis of Lewy body disease. PRKG1, MAPK13, andGAK are negative regulators of the phosphorylation of alpha-synuclein.Thus, agents that activate these kinases decrease phosphorylation ofalpha-synuclein and can be used in treatment or prophylaxis of Lewy bodydisease.

GRK6, also called GPRK6, is a G protein-coupled receptor kinase and isinvolved in signal transduction. G protein-coupled receptor kinasesphosphorylate and desensitize ligand-activated G protein-coupledreceptors. GRK6 expression has previously been shown to be significantlyelevated in the MPTP-lesioned group in most brain regions. For thepurposes of consistency, the name GRK6 will be used throughout thepresent patent application. An exemplary accession number is provided inTable 1A. The sequence for GRK6 can be found in any one of Teli, et al.,Anesthesiology 98 (2), 343-348 (2003); Miyagawa, et al., Biophys. Res.Commun. 300 (3), 669-673 (2003); Gaudreau, et al., J. Biol. Chem. 277(35), 31567-31576 (2002); Grange-Midroit, et al., Brain Res. Mol BrainRes. 101 (1-2), 39-51 (2002); Willets, et al., J. Biol. Chem. 277 (18),15523-15529 (2002); Blaukat, et al., J. Biol. Chem. 276 (44),40431-40440 (2001); Zhou, et al., J. Pharmacol. Exp. Ther. 298 (3),1243-1251 (2001); Pronin, et al., J. Biol. Chem. 275 (34), 26515-26522(2000); Tiruppathi, Proc. Natl. Acad. Sci. U.S.A., 97 (13), 7440-7445(2000); Premont, et al. J. Biol. Chem., 274 (41), 29381-29389 (1999);Brenninkmeijer, et al., J. Endocrinol. 162 (3), 401-408 (1999); Hall, etal., J. Biol. Chem. 274 (34), 24328-24334 (1999); Lazari, et al., Mol.Endocrinol. 13 (6), 866-878 (1999); Milcent, et al., Biochem. Biophys.Res. Commun. 259 (1), 224-229 (1999); Premont, Proc. Natl. Acad. Sci.U.S.A. 95 (24), 14082-14087 (1998); Stoffel, et al., Biochemistry 37(46), 16053-16059 (1998); Loudon, et al., J. Biol. Chem. 272 (43),27422-27427 (1997); Freedman, et al., J. Biol. Chem. 272 (28),17734-17743 (1997); Bullrich, et al., Cytogenet. Cell Genet. 70 (3-4),250-254 (1995); Stoffel, et al.; J. Biol. Chem. 269 (45), 27791-27794(1994); Loudon, et al., J. Biol. Chem. 269 (36), 22691-22697 (1994);Haribabu and Snyderman, Proc. Natl. Acad. Sci. U.S.A. 90 (20), 9398-9402(1993); and Benovic and Gomez, J. Biol. Chem. 268 (26), 19521-19527(1993); reference to the amino acid or nucleic acid sequence of GRK6includes the amino acid or nucleic acid sequence of any of thesereferences and allelic variants thereof.

Casein kinase 2 (also called Casein kinase II, CKII, CSNK2 and CSNKII)has been reported to phosphorylate alpha-synuclein. For consistency, thename CKII will be used herein. The sequence for CKII has been providedin Genbank under the following accession numbers: NM_(—)001896,NM_(—)001320 and/or can be found in any one of: Panasyu, et al. J. Biol.Chem. 281 (42), 31188-31201 (2006); Salvi, et al. FEBS Lett. 580 (16),3948-3952 (2006); Lim et al. Cell 125 (4), 801-814 (2006); Llorens, etal. Biochem. J. 394 (Pt. 1), 227-236, (2006); Bjorling-Poulsen, et al.Oncogene 24 (40), 6194-6200 (2005); Schubert, et al. Eur. J. Biochem.204 (2), 875-883 (1991); Voss, et al. J. Biol. Chem. 266 (21),13706-13711 (1991); Yang-Feng, et al. Genomics 8 (4), 741-742 (1990);Heller-Harrison, et al. Biochemistry 28 (23), 9053-9058 (1989);Ackermann, et al. Mol. Cell. Biochem. 274 (1-2), 91-101 (2005);Barrios-Rodiles, et al. Science 307 (5715), 1621-1625 (2005); Andersen,et al. Nature 433 (7021), 77-83 (2005); Ballif, et al. Mol. Cell.Proteomics, 3 (11), 1093-1101 (2004); Beausoleil, et al. PNAS, USA 101(33), 12130-12135 (2004); Marais, et al. EMBO J. 11 (1), 97-105 (1992).Reference to the amino acid or nucleic acid sequence of CKII includesthe amino acid or nucleic acid sequence of any of these references andallelic variants thereof.

IKBKB and the related IKBKA are positive regulators of the NFkBinflammatory pathway. The sequence for IKBKB has been provided inGenbank under the following accession number: NM_(—)001556 and/or can befound in any one of: Caterino, et al. FEBS Lett. 580 (28-29), 6527-6532(2006); Castle, et al. Genome Biol. 4 (10), R66 (2003); Satoh, et al.Biochim, Biophys. Acta 1600 (103), 61-67 (2002), Caohuy, and Pollard, J.Biol. Chem. 277 (28), 25217-25225 (2002); Yu, et al. J. Biol. Chem. 277(18), 15819-15827 (2002); Selbert, et al. J. Cell. Sci. 108 (Pt.1),85095 (1995); Shirvan, et al. Biochemistry 33 (22), 6888-6901 (1994);Creutz, et al. Biochem. Biophys. Res. Commun. 184 (1), 347-352 (1992);Megendzo, et al. J. Biol. Chem. 266 (5), 3228-3232 (1991); Burns, et al.PNAS, USA 86 (10), 3798-3802 (1989). Reference to the amino acid ornucleic acid sequence of IKBKB includes the amino acid or nucleic acidsequence of any of these references and allelic variants thereof.

Synphilin is a synuclein-associated protein that has been shown to bindalpha-synuclein. Although not a kinase itself, Synphilin was foundherein to promote phosphorylation of synuclein particularly incombination with PLK2. The synphilin appeared to promote phosphorylationof synuclein in a PLK2-dependent manner. The sequence for synphilin hasbeen provided in Genbank under the following accession number:NM_(—)005460 and/or can be found in any one of: Tanji, et al. Am. J.Pathol. 169 (2), 553-565 (2006); Eyal, et al. PNAS, USA 103 (15),5917-5922 (2006); Avraham, et al. J. Biol. Chem. 280 (52), 42877-42886(2005); Bandopadhyay, et al. Neurobiol. Dis. 20 (2), 401-411 (2005); Limet al. J. Neurosci. 25 (8), 2002-2009 (2005); Ribeiro, et al. J. Biol.Chem. 277 (26), 23927-23933 (2002); Chung, et al. Nat. Med. 7 (10),1144-1150 (2001); Engelender, et al. Mamm. Genome 11 (9), 763-766(2000); Engelender, et al. Nat. Genet. 22 (1), 110-114 (1999). Referenceto the amino acid or nucleic acid sequence of synphilin includes theamino acid or nucleic acid sequence of any of these references andallelic variants thereof

V. Agents to Modulate Synuclein Kinase Activity or Expression

In one aspect, the invention provides methods of effecting treatment orprophylaxis of an LBD by administering an agent that modulates activityor expression of a kinase described herein. A number of agents ofwell-characterized general classes can be used. Without limitation theseinclude inhibitory nucleic acids (e.g., siRNA, antisense RNA,ribozymes), inhibitory proteins (e.g., zinc finger proteins),antibodies, and small molecule inhibitors.

Preferably the gene to be inhibited is PLK2 or GRK6 because the kinasesencoded by these genes directly phosphorylate alpha-synuclein, andparticularly PLK2. APEG1, CDC7L1, MET GRK1, GRK2, GRK6, IKBKB, CKII andGRK7 genes are also preferred targets for inhibition because they arelikely to encode indirect activators of the direct kinase(s). PRKG1,MAPK13, and GAK are preferred candidates for activation in Lewy bodydiseases because they encode negative regulators of the phosphorylationof alpha-synuclein. The synphilin gene is a preferred target forinhibition because, although synphilin is not a kinase, it is associatedwith increased phosphorylation of alpha-synuclein (typically in thepresence of a kinase such as PLK2).

Inhibitors that show specificity for PLK2 over one or more otherpolo-like kinase family members (i.e., PLK1, PLK3, and PLK4) arepreferred. Inhibitors especially suited for therapeutic use may beidentified by selecting for at least one of the following properties:

-   -   I. Inhibits PLK2 activity and has no, or reduced, effect on        PLK1.    -   II. Inhibits PLK2 activity and has no, or reduced, effect on        PLK3.    -   III. Inhibits PLK2 activity and has no, or reduced, effect on        PLK4.    -   IV. Inhibits PLK2 activity and has no, or reduced, effect on        PLK1 and PLK3.    -   V. Inhibits PLK2 activity and has no, or reduced, effect on PLK1        and PLK4.    -   VI. Inhibits PLK2 activity and has no, or reduced, effect on        PLK1, PLK3 and PLK4.

As used in this context, “no effect” means administration of the agentdoes not reduce expression, or reduces expression by a physiologicallyinsignificant degree. “Reduced effect” means that the EC₅₀ or K_(i)values for inhibiting PLK2 is lower than the EC₅₀ for the referencePLK(s). In some embodiments the EC₅₀ may be at least 2-fold lower, andis sometimes at least 10-fold lower, and may be at least 100-fold, oreven at least 1000-fold lower.

As used in this context, inhibition of PLK2 “activity” can result fromreducing protein expression (e.g., reducing expression of the PLK2 gene,interfering with processing of PLK2 RNA, reducing the half-life of PLK2mRNA or protein) or by competitive or noncompetitive inhibition of thePLK2 kinase activity.

Inhibitors that show specificity for PLK2 over non-polo kinases,especially other kinases expressed in the tissues to which the agent isdelivered are especially preferred. In preferred embodiments the agentdoes not inhibit non-polo kinases (or has a EC₅₀ greater than 10-timeshigher, sometimes 100-times greater, and sometimes greater than1000-times higher) for non-polo kinases compared to PLK2. However,inhibition of other kinases may be tolerated depending on the role andexpression of the kinase. For example, a kinase that functions in thegut may not be affected by an inhibitor delivered to the brain.

To further guide the reader, inhibitory nucleic acids (e.g., siRNA,antisense RNA, ribozymes), inhibitory proteins (e.g., zinc fingerproteins), antibodies, and small molecule inhibitors are discussedbelow.

A. Inhibitory Polynucleotides

Several examples of inhibitors of target kinases, including PLK2, aredescribed below. Polynucleotide inhibitors are designed to bind specifictarget sequences within a target transcript. Preferably the inhibitorsbind to a target site in a PLK2 RNA without binding a target site in:PLK1 and/or PLK3 and/or PLK4. Suitable target sites are identified byselecting segments of PLK2 that have no exact corresponding segment inother PLKs. Preferably, a selected segment of PLK2 lacks a correspondingsegment having substantial sequence identity (for example, a selectedsegment from PLK2 should show less than 95, 90, 75 or 50% sequenceidentity with the closest corresponding segment in PLK4). A selectedtarget segment is also preferably screened against a gene database toensure that it does not show significant sequence identity withunrelated genes by chance.

Polynucleotide inhibitors of PLK2 preferably show at least 30, 50, 75,95, or 99% inhibition of levels of PLK2 mRNA or protein with little orno detectable reduction in levels of PLK1 and/or PLK3 and/or PLK4 mRNAor protein (i.e, less than 10, 5 or 1% inhibition). Protein expressioncan be quantified by forming immunological analyses using an antibodythat specifically binds to the protein followed by detection of complexformed between the antibody and protein. mRNA levels can be quantifiedby, for example, dot blot analysis, in-situ hybridization, RT-PCR,quantitative reverse-transcription PCR (i.e., the so-called “TaqMan”methods), Northern blots and nucleic acid probe array methods.

i) Short Inhibitory RNAs

siRNAs are relatively short, at least partly double stranded, RNAmolecules that serve to inhibit expression or translation of acomplementary mRNA transcript, such as a kinase transcript. Although anunderstanding of mechanism is not required for practice of theinvention, it is believed that siRNAs act by inducing degradation of acomplementary mRNA transcript. Principles for design and use of siRNAsgenerally are described by WO 99/32619, Elbashir, EMBO J. 20, 6877-6888(2001) and Nykanen et al., Cell 107, 309-321 (2001); WO 01/29058. siRNAsare formed from two strands of at least partly complementary RNA, eachstrand preferably of 10-30, 15-25, or 17-23 or 19-21 nucleotides long.The strands can be perfectly complementary to each other throughouttheir length or can have single stranded 3′-overhangs at one or bothends of an otherwise double stranded molecule. Single strandedoverhangs, if present, are usually of 1-6 bases with 1 or 2 bases beingpreferred. The antisense strand of an siRNA is selected to besubstantially complementary (e.g., at least 80, 90, 95% and preferably100% complementary) to a segment of a transcript from a gene of theinvention. Any mismatched bases preferably occur at or near the ends ofthe strands of the siRNA. Mismatched bases at the ends can bedeoxyribonucleotides. The sense strand of an siRNA shows an analogousrelationship with the complement of the segment of the gene transcriptof interest. siRNAs having two strands, each having 19 bases of perfectcomplementarity, and having two unmatched bases at the 3′ end of thesense strand and one at the 3′ end of the antisense strand areparticularly suitable.

If an siRNA is to be administered as such, as distinct from the form ofDNA encoding the siRNA, then the strands of an siRNA can contain one ormore nucleotide analogs. The nucleotide analogs are located at positionsat which inhibitor activity is not substantially affected, e.g. in aregion at the 5′-end and/or the 3′-end, particularly single strandedoverhang regions. Preferred nucleotide analogues are sugar- orbackbone-modified ribonucleotides. Nucleobase-modified ribonucleotides,i.e. ribonucleotides, containing a non-naturally occurring nucleobaseinstead of a naturally occurring nucleobase such as uridines orcytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine,5-bromo uridine; adenosines and guanosines modified at the 8 position,e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g. N6-methyl adenosine are also suitable.In preferred sugar-modified ribonucleotides, the 2′ OH-group is replacedby a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN,wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g. of phosphothioate group. A further preferred modification is tointroduce a phosphate group on the 5′ hydroxide residue of an siRNA.Such a group can be introduced by treatment of an siRNA with ATP and T4kinase. The phosphodiester linkages of natural RNA can also be modifiedto include at least one of a nitrogen or sulfur heteroatom.Modifications in RNA structure can be tailored to allow specific geneticinhibition while avoiding a general panic response in some organismswhich is generated by dsRNA. Likewise, bases can be modified to blockthe activity of adenosine deaminase.

One example of such an agent is siRNA specific for PLK2. siRNAs to thegene encoding PLK2 can be specifically designed using methods describedbelow. An exemplary accession number for PLK2 is [NM_(—)006622] asprovided in Table 1A. The amino acid sequence of human PLK2 is also setforth as SEQ ID NO:2 and SEQ ID NO:1 is the nucleic acid sequenceencoding the amino acid sequence. For convenience exemplary sequencesare provided below.

Human PLK2 (SEQ ID NO:1)    1 gcacaagtgg accggggtgt tgggtgctagtcggcaccag aggcaagggt gcgaggacca   61 cggccggctc ggacgtgtga ccgcgcctagggggtggcag cgggcagtgc ggggcggcaa  121 ggcgaccatg gagcttttgc ggactatcacctaccagcca gccgccagca ccaaaatgtg  181 cgagcaggcg ctgggcaagg gttgcggagcggactcgaag aagaagcggc cgccgcagcc  241 ccccgaggaa tcgcagccac ctcagtcccaggcgcaagtg cccccggcgg cccctcacca  301 ccatcaccac cattcgcact cggggccggagatctcgcgg attatcgtcg accccacgac  361 tgggaagcgc tactgccggg gcaaagtgctgggaaagggt ggctttgcaa aatgttacga  421 gatgacagat ttgacaaata acaaagtctacgccgcaaaa attattcctc acagcagagt  481 agctaaacct catcaaaggg aaaagattgacaaagaaata gagcttcaca gaattcttca  541 tcataagcat gtagtgcagt tttaccactacttcgaggac aaagaaaaca tttacattct  601 cttggaatac tgcagtagaa ggtcaatggctcatattttg aaagcaagaa aggtgttgac  661 agagccagaa gttcgatact acctcaggcagattgtgtct ggactgaaat accttcatga  721 acaagaaatc ttgcacagag atctcaaactagggaacttt tttattaatg aagccatgga  781 actaaaagtt ggggacttcg gtctggcagccaggctagaa cccttggaac acagaaggag  841 aacgatatgt ggtaccccaa attatctctctcctgaagtc ctcaacaaac aaggacatgg  901 ctgtgaatca gacatttggg ccctgggctgtgtaatgtat acaatgttac tagggaggcc  961 cccatttgaa actacaaatc tcaaagaaacttataggtgc ataagggaag caaggtatac 1021 aatgccgtcc tcattgctgg ctcctgccaagcacttaatt gctagtatgt tgtccaaaaa 1081 cccagaggat cgtcccagtt tggatgacatcattcgacat gacttttttt tgcagggctt 1141 cactccggac agactgtctt ctagctgttgtcatacagtt ccagatttcc acttatcaag 1201 cccagctaag aatttcttta agaaagcagctgctgctctt tttggtggca aaaaagacaa 1261 agcaagatat attgacacac ataatagagtgtctaaagaa gatgaagaca tctacaagct 1321 taggcatgat ttgaaaaaga cttcaataactcagcaaccc agcaaacaca ggacagatga 1381 ggagctccag ccacctacca ccacagttgccaggtctgga acacccgcag tagaaaacaa 1441 gcagcagatt ggggatgcta ttcggatgatagtcagaggg actcttggca gctgtagcag 1501 cagcagtgaa tgccttgaag acagtaccatgggaagtgtt gcagacacag tggcaagggt 1561 tcttcgggga tgtctggaaa acatgccggaagctgattgc attcccaaag agcagctgag 1621 cacatcattt cagtgggtca ccaaatgggttgattactct aacaaatatg gctttgggta 1681 ccagctctca gaccacaccg tcggtgtccttttcaacaat ggtgctcaca tgagcctcct 1741 tccagacaaa aaaacagttc actattacgcagagcttggc caatgctcag ttttcccagc 1801 aacagatgct cctgagcaat ttattagtcaagtgacggtg ctgaaatact tttctcatta 1861 catggaggag aacctcatgg atggtggagatctgcctagt gttactgata ttcgaagacc 1921 tcggctctac ctccttcagt ggctaaaatctgataaggcc ctaatgatgc tctttaatga 1981 tggcaccttt caggtgaatt tctaccatgatcatacaaaa atcatcatct gtagccaaaa 2041 tgaagaatac cttctcacct acatcaatgaggataggata tctacaactt tcaggctgac 2101 aactctgctg atgtctggct gttcatcagaattaaaaaat cgaatggaat atgccctgaa 2161 catgctctta caaagatgta actgaaagacttttcgaatg gaccctatgg gactcctctt 2221 ttccactgtg agatctacag ggaagccaaaagaatgatct agagtatgtt gaagaagatg 2281 gacatgtggt ggtacgaaaa caattcccctgtggcctgct ggactggttg gaaccagaac 2341 aggctaaggc atacagttct tgactttggacaatccaaga gtgaaccaga atgcagtttt 2401 ccttgagata cctgttttaa aaggtttttcagacaatttt gcagaaaggt gcattgattc 2461 ttaaattctc tctgttgaga gcatttcagccagaggactt tggaactgtg aatatacttc 2521 ctgaagggga gggagaaggg aggaagctcccatgttgttt aaaggctgta attggagcag 2581 cttttggctg cgtaactgtg aactatggccatatataatt ttttttcatt aatttttgaa 2641 gatacttgtg gctggaaaag tgcattccttgttaataaac tttttattta ttacagccca 2701 aagagcagta tttattatca aaatgtctttttttttatgt tgaccatttt aaaccgttgg 2761 caataaagag tatgaaaacg cagaaaaaaaaaaaa Human PLK2 (SEQ ID NO:2)MELLRTITYQPAASTKMCSQALGKGCGADSKKKRPPQPPEESQPPQSQAQVPPAAPHHHHHHSHSGPEISRIIVDPTTGKRYCRGKVLGKGGFAKCYEMTDLTNNKVYAAKIIPHSRVAKPHQREKIDKEIELHRILHHKHVVQFYHYFEDKENIYILLEYCSRRSMAHILKARKVLTEPEVRYYLRQIVSGLKYLHEQEILHRDLKLGNFFINEAMELKVGDFGLAARLEPLEHRRRTICGTPNYLSPEVLNKQGHGCESDIWALGCVMYTMLLGRPPFETTNLKETYRCIREARYTMPSSLLAPAKHLIASMLSKNPEDRPSLDDIIRHDFFLQGFTPDRLSSSCCHTVPDFHLSSPAKNFFKKAAAALFGGKKDKARYIDTHNRVSKEDEDIYKLRHDLKKTSITQQPSKHRTDEELQPPTTTVARSGTPAVENKQQIGDAIRMIVRGTLGSCSSSSECLEDSTMGSVADTVARVLRGCLENMPEADCIPKEQLSTSFQWVTKWVDYSNKYGFGYQLSDHTVGVLFNNGAHMSLLPDKKTVHYYAELGQCSVFPATDAPEQFISQVTVLKYFSHYMEENLMDGGDLPSVTDIRRPRLYLLQWLKSDKALMMLFNDGTFQVNFYHDHTKIIICSQNEEYLLTYINEDRISTTFRLTTLLMSGCSSELKNRMEYALNMLLQRCN

ii) Design and Production of siRNA

An advantage of inhibitory polynucleotides is that they can be designedto be highly target specific. For example, siRNAs specific for PLK2 canbe designed using target sequences that distinguish PLK2 from otherPLKs. The program “siDESIGN” (Dharmacon, Inc., Lafayette, Colo.) can beused to predict siRNA sequences for any nucleic acid sequence, and isavailable on the World Wide Web at dharmacon.com. A number of otherprograms for designing siRNAs are available from others, includingGenscript (available on the Web at genscript.com/ssl-bin/app/mai) andfrom the Whitehead Institute for Biomedical Researchjura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/. Guidelines fordesigning siRNA are available in the scientific literature (see, e.g.,Elbashir et al., 2001, “Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells” Nature. 411:494-8.; andElbashir et al., 2001, “RNA interference is mediated by 21- and22-nucleotide RNAs” Genes Dev. 15:188-200) and published on the Web(see, e.g., “maiweb.com/RNAi/siRNA Design/” and“protocol-online.org/prot/Protocols/Rules-of-siRNA-design-for-RNA-interference-RNAi-3210.html”).

There are a variety of ways to produce siRNAs. siRNAs can be generatedusing kits which generate siRNA from the kinase (e.g., PLK2) gene. Forexample, the “Dicer siRNA Generation” kit (catalog number T510001, GeneTherapy Systems, Inc., San Diego, Calif.) uses the recombinant humanenzyme “dicer” in vitro to cleave long double stranded RNA into 22 bpsiRNAs. By producing a mixture of siRNAs, the kit permits a high degreeof success in generating siRNAs that will reduce expression of thetarget gene. Similarly, the Silencer™ siRNA Cocktail Kit (RNase III)(catalog no. 1625, Ambion, Inc., Austin, Tex.) generates a mixture ofsiRNAs from dsRNA using RNase III instead of dicer. Like dicer, RNaseIII cleaves dsRNA into 12-30 bp dsRNA fragments with 2 to 3 nucleotide3′ overhangs, and 5′-phosphate and 3′-hydroxyl termini. According to themanufacturer, dsRNA is produced using T7 RNA polymerase, and reactionand purification components included in the kit. The dsRNA is thendigested by RNase III to create a population of siRNAs. The kit includesreagents to synthesize long dsRNAs by in vitro transcription and todigest those dsRNAs into siRNA-like molecules using RNase III. Themanufacturer indicates that the user need only supply a DNA templatewith opposing T7 phage polymerase promoters or two separate templateswith promoters on opposite ends of the region to be transcribed.

The siRNAs of the invention can also be expressed from vectors.Typically, such vectors are administered in conjunction with a secondvector encoding the corresponding complementary strand. Once expressed,the two strands anneal to each other and form the functional doublestranded siRNA. One exemplar vector suitable for use in the invention ispSuper, available from OligoEngine, Inc. (Seattle, Wash.). In someembodiments, the vector contains two promoters, one positioneddownstream of the first and in antiparallel orientation. The firstpromoter is transcribed in one direction, and the second in thedirection antiparallel to the first, resulting in expression of thecomplementary strands. In yet another set of embodiments, the promoteris followed by a first segment encoding the first strand, and a secondsegment encoding the second strand. The second strand is complementaryto the palindrome of the first strand. Between the first and the secondstrands is a section of RNA serving as a linker (sometimes called a“spacer”) to permit the second strand to bend around and anneal to thefirst strand, in a configuration known as a “hairpin.”

The formation of hairpin RNAs, including use of linker sections, is wellknown in the art. Typically, an siRNA expression cassette is employed,using a Polymerase III promoter such as human U6, mouse U6, or human H1.The coding sequence is typically a 19-nucleotide sense siRNA sequencelinked to its reverse complementary antisense siRNA sequence by a shortspacer. Nine-nucleotide spacers are typical, although other spacers canbe designed. Further, 5-6 T's are often added to the 3′ end of theoligonucleotide to serve as a termination site for Polymerase III. Seealso, Yu et al., Mol Ther 7(2):228-36 (2003); Matsukura et al., NucleicAcids Res 31(15):e77 (2003).

The siRNA targets can be targeted by hairpin siRNA as follows. To attackthe same targets by short hairpin RNAs, produced by a vector (permanentRNAi effect), sense and antisense strand can be put in a row with a loopforming sequence in between and suitable sequences for an adequateexpression vector to both ends of the sequence.

The siRNA of the invention can be made using any suitable method forproducing a nucleic acid, such as the chemical synthesis and recombinantmethods disclosed herein and known to one of skill in the art. It willbe appreciated that the oligonucleotides can be made using nonstandardbases (e.g., other than adenine, cytidine, guanine, and uridine) ornonstandard backbone structures to provide desirable properties (e.g.,increased nuclease-resistance, tighter-binding, stability or a desiredTm). Techniques for rendering oligonucleotides nuclease-resistantinclude those described in PCT Publication WO 94/12633. A wide varietyof useful modified oligonucleotides may be produced, includingoligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsenet al., 1991, Science 254:1497) or incorporating 2′-O-methylribonucleotides, phosphorothioate nucleotides, methyl phosphonatenucleotides, phosphotriester nucleotides, phosphorothioate nucleotides,phosphoramidates.

For example and not limitation, the following are examples of siRNAsequences that can be used to inhibit PLK2. “Start position” refers tothe sequence at accession number NM_(—)006622.

Sense Strand Sequence Region Start Pos  1 CCGGAGATCTCGCGGATTA ORF 326  2GGGGCAAAGTGCTGGGAAA ORF 378  3 TCACAGCAGAGTAGCTAAA ORF 469  4GGGAAAAGATTGACAAAGA ORF 498  5 GATTGTGTCTGGACTGAAA ORF 691  6GCACAGAGATCTCAAACTA ORF 733  7 ACACAGAAGGAGAACGATA ORF 829  8AGGAGAACGATATGTGGTA ORF 836  9 CATAAGGGAAGCAAGGTAT ORF 1000 10GCTAGTATGTTGTCCAAAA ORF 1061 11 GAAGACATCTACAAGCTTA ORF 1304 12CATCAATGAGGATAGGATA ORF 2062 13 GACATGTGGTGGTACGAAA 3′ UTR 2281 14CAGAACAGGCTAAGGCATA 3′ UTR 2335 15 GTGCATTCCTTGTTAATAA 3′ UTR 2660

The siRNAs above were designed using the siDESIGN® center at wwwfollowed by dharmacon.com/DesignCenter/DesignCenterPage.aspx. Each willbe double stranded and have a “TT” overhang.

As additional examples, siRNAs from the Ambion Kinase siRNA Library(Ambion, Austin, Tex.) can be used to inhibit PLK2. Exemplary sequencesare provided below. Each siRNA is double-stranded with the final TT's(present on both strands) as overhangs:

16. GGUAUACAAUGCCGUCCUCTT 17. GGACUUUGGAACUGUGAAUTT 18.GGGAAAAGAUUGACAAAGATT

iii) Antisense Polynucleotides

Antisense polynucleotides can cause suppression by binding to, andinterfering with the translation of sense mRNA, interfering withtranscription, interfering with processing or localization of RNAprecursors, repressing transcription of mRNA or acting through someother mechanism (see, e.g., Sallenger et al. Nature 418, 252 (2002). Theparticular mechanism by which the antisense molecule reduces expressionis not critical. Typically antisense polynucleotides comprise asingle-stranded antisense sequence of at least 7 to 10 to typically 20or more nucleotides that specifically hybridize to a sequence from mRNAof a kinase gene of the invention. Some antisense polynucleotides arefrom about 10 to about 50 nucleotides in length or from about 14 toabout 35 nucleotides in length. Some antisense polynucleotides arepolynucleotides of less than about 100 nucleotides or less than about200 nucleotides. In general, the antisense polynucleotide should be longenough to form a stable duplex but short enough, depending on the modeof delivery, to administer in vivo, if desired. The minimum length of apolynucleotide required for specific hybridization to a target sequencedepends on several factors, such as G/C content, positioning ofmismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone, peptidenucleic acid, phosphorothioate), among other factors.

iv) Ribozymes

Ribozymes are RNA molecules that act as enzymes and can be engineered tocleave other RNA molecules at specific sites. The ribozyme itself is notconsumed in this process, and can act catalytically to cleave multiplecopies of mRNA target molecules. General rules for the design ofribozymes that cleave target RNA in trans are described in Haseloff &Gerlach, (1988) Nature 334:585-591 and Hollenbeck, (1987) Nature328:596-603 and U.S. Pat. No. 5,496,698. Ribozymes typically include twoflanking segments that show complementarity to and bind to two sites ona transcript (target subsites) of a gene encoding a kinase of theinvention and a catalytic region between the flanking segments. Theflanking segments are typically 5-9 nucleotides long and optimally 6 to8 nucleotides long. The catalytic region of the ribozyme is generallyabout 22 nucleotides in length. The mRNA target contains a consensuscleavage site between the target subsites having the general formulaNUN, and preferably GUC. (Kashani-Sabet and Scanlon, (1995) Cancer GeneTherapy 2:213-223; Perriman, et al., (1992) Gene (Amst.) 113:157-163;Ruffner, et al., (1990) Biochemistry 29: 10695-10702); Birikh, et al.,(1997) Eur. J. Biochem. 245:1-16; Perrealt, et al., (1991) Biochemistry30:4020-4025). The specificity of a ribozyme can be controlled byselection of the target subsites and thus the flanking segments of theribozyme that are complementary to such subsites. Ribozymes can bedelivered either as RNA molecules or in the form of DNA encoding theribozyme as a component of a replicable vector or in nonreplicable formas described below.

Expression of a target kinase gene can also be reduced by deliveringnucleic acids having sequences complementary to the regulatory region ofthe target gene (i.e., the target gene promoter and/or enhancers) toform triple helical structures which prevent transcription of the targetgene in target cells in the body. See generally, Helene, (1991),Anticancer Drug Des., 6(6):569-584; Helene, et al., (1992), Ann. N.Y.Acad. Sci., 60:27-36; and Maher, (1992), Bioassays 14(12):807-815.

v) Administration of siRNA and Other Inhibitory Nucleic Acids

The brain is the therapeutic target of kinase inhibitors of theinvention. Therapeutic polynucleotides such as siRNAs, ribozymes andantisense polynucleotides can be administered in a number of ways.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion, by bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oral,rectal and intestinal mucosa, etc.), and may be administered togetherwith other biologically active agents. In general, therapeuticpolynucleotides can be administered remotely (e.g., by i.v. injection)with a carrier that facilitates transfer to the brain or they can bedelivered directly to the brain.

For direct administration to the brain, siRNAs (i.e., pharmaceuticalcompositions containing siRNAs) can be administered by any suitableroute, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir, direct injection or perfusion at the lesion site, injectioninto the brain arterial system, or by chemical or osmotic opening of theblood-brain barrier.

In one approach the therapeutic polynucleotide (e.g., siRNA) isdelivered as a peptide conjugate. Kumar et al. exploited the fact thatneurotropic viruses, such as rabies viruses, that preferentially infectthe nervous system can penetrate the brain. The rabies virus achievesthis through glycoprotein on its lipid envelope. To transfer siRNA tothe neural cells in the brain, Kumar et al. identified a 29-residuepeptide from the rabies virus glycoprotein (RVG) envelope thatselectively binds to the acetylcholine receptor expressed by neurons.They fused this peptide with a sequence of 9 arginine residues thatbinds to siRNAs. After intravaneous injection into mice with thispeptide-conjugated siRNA, they found that the peptide not only enabledthe transvascular delivery of siRNA to the brain but also resulted inefficient gene silencing (Kumar et al., (2007) Nature 448:39-43).

The therapeutic polynucleotide (e.g., siRNA) can be delivered using aliposome and targeted monoclonal antibody system. Pardridge reportedthat chemically modified liposomes conjugated to monoclonal antibodiesraised against epidermal growth factor can penetrate mouse brain.Plasmid DNA encoding for short hairpin RNA (shRNA) was delivered to thebrain following intravenous administration with pegylatedimmunoliposomes (PILs). The plasmid DNA is encapsulated in liposome,which is pegylated, and conjugated with receptor specific targetingmonoclonal antibodies. Intravenous RNAi with PILs enables a 90%knockdown of the human epidermal growth factor receptor, which resultsin a 90% increase in survival time in mice with intra-cranial braincancer (Pardridge, (2007) Adv. Drug Deliv. Rev. 59:141-152).

Similarly, Boado disclosed the use of the “Trojan Horse Liposome” (THL)technology as an effective delivery system to deliver shRNA to thebrain. The tissue target specificity of THL is given by conjugation ofapproximately 1% of the PEG residues in the liposome to peptidomimeticmonoclonal antibodies that bind to specific endogenous receptors (i.e.insulin and transferrin receptors) located on both the BBB and the braincellular membranes, respectively. (Boado (2007) Pharm. Res.24:1772-1787).

The therapeutic polynucleotide (e.g., siRNA) can be delivered by thecombined use of receptor specific antibody delivery systems andavidin-biotin technology. The siRNA was mono-biotinylated on eitherterminus of the sense strand, in parallel with the production of aconjugate of the targeting monoclonal antibody and streptavidin. Ratglial cells permanently transfected with the luciferase gene wereimplanted in the brain of adult rats. Following the formation ofintra-cranial tumors, the rats were treated with a single intravenousinjection of biotinylated siRNA attached to a transferrin receptorantibody via a biotin-streptavidin linker. The intravenousadministration of the siRNA caused a 69-81% decrease in luciferase geneexpression in the intracranial brain cancer in vivo (Xia et al., (2007)Pharm. Res. 24:2309-2316).

The therapeutic polynucleotide (e.g., siRNA) can be delivered bystereotactic surgery or injection. Davidson and Boudreau reviewed intheir article that siRNA can be delivered into the brain usingneurosurgical method of stereotaxis and showed that a decrease in thetranscription of certain genes alleviated symptoms of neuronal diseases(Davidson and Boudreau, (2007) Neuron 53:781-788).

Xia et al. reported that upon intracerebellar injection, recombinantadeno-associated virus vectors expressing short hairpin RNAs, which onceexpressed are processed into siRNAs, improved motor coordination,restored cerebellar morphology and resolved characteristic ataxin-1inclusions in Purkinje cells of spinocerebellar ataxia type 1 mice (Xiaet al., (2004) Nature Med. 10:816-820).

Further, DiFiglia et al. reported injecting intrastriatallycholesterol-conjugated siRNA that targets human huntingtin mRNA. Theauthors found that a single administration into the adult mouse striatumof the siRNA effected silencing of the gene, attenuated neuronalpathology, and delayed the abnormal behavioral phenotype observed in arapid-onset, viral transgenic mouse model of Huntington's disease(DiFiglia et al. (2007) Proc. Natl. Acad. Sci. USA 104:17204-17209). Itis noted that such method results only in localized delivery around theinjection site, with no widespread effect within the brain.

Singer et al. disclosed using modified lentiviral vectors to deliversiRNAs into the brain cells of the transgenic mice that were producingvast amounts of human beta-amyloid and whose brains where littered withplaques. They found that lentiviral vector delivery of beta-secretasesiRNA specifically reduced the cleavage of amyloid precursor protein andneurodegeneration in vivo and indicated that this approach could havepotential therapeutic value for treatment of Alzheimer disease (Singeret al. (2005) Nature Neurosci. 8:1343-1349; reviewed in Orlacchio et al.(2007) Mini. Rev. Med. Chem. 7:1166-1176).

Koutsilieri et al. reviewed the literature in the field of siRNA,disclosed different siRNA target strategies aiming for anallele-specific degradation of disease-inducing mRNA and its applicationin animal models of neurodegenerative diseases, including Alzheimer'sdisease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease(HD) and spinocerebellar ataxia (SCA1) (Koutsilieri et al. (2007) J.Neural Transm Suppl. (72):43-49).

Hassani et al. demonstrated that cationic lipids and polyethylenimine(PEI) based polyplexes provided efficient delivery of siRNAs into thebrains of new born mice, producing >80% inhibition of an exogenous genewith only picomolar levels of siRNA (Hassani et al. (2005) J. Gene Med.7:198-207).

Kateb et al. employed nanotechnology as a method for delivering drugs tothe brain for treatment of brain cancers. Specifically, the authorsdisclosed the use Multi-Walled Carbon Nanotubes (MWCNTs) as nanovectorsfor transporting siRNA (Kateb et al. (2007) Neuroimage 37 Suppl1:S9-17).

The therapeutic polynucleotide (e.g., siRNA) can be used in combinationwith other agents to improve or enhance the therapeutic effect ofeither. This process can involve administering both agents to thepatient at the same time, either as a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, wherein onecomposition includes the siRNA of the invention and the other includesthe second agent(s). The siRNA therapy also can precede or follow theother agent treatment by intervals ranging from minutes to weeks.

Polynucleotides can be delivered via a controlled release system. As anexample, a pump may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:201-240; Buchwald et al., 1980, Surgery 88:507-516;Saudek et al., 1989, N. Engl. J. Med. 321:574-579). Alternatively,polymeric materials can be used (Medical Applications of ControlledRelease, Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974;Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball, eds., Wiley, N.Y., 1984; Ranger and Peppas, 1983,Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science228:190-192; During et al., 1989, Ann. Neurol. 25:351-356; Howard etal., 1989, J. Neurosurg. 71:858-863). In yet another alternative, acontrolled release system can be placed in proximity of the therapeutictarget, i.e., the brain, thus requiring only a fraction of the systemicdose (e.g., Goodson, 1984, In: Medical Applications of ControlledRelease, supra, Vol. 2, pp. 115-138). Other controlled release systemsare discussed in the review by Langer (1990, Science 249:1527-1533).

Other approaches can include the use of various transport and carriersystems, for example though the use of viral and/or non-viral deliverysystems. For example, siRNA can be introduced into the brain in a virusmodified to serve as a vehicle without causing pathogenicity. The viruscan be, e.g., adenovirus, fowlpox virus, vaccinia virus, lentivirus, orneurotropic virus such as HIV, herpes simplex virus, flavivirus, orrabies virus. (Li et al., Methods Mol. Biol. 309:261-272 (2005);Davidson et al., Neuron 53:781-788 (2007); Xia et al., Nature Med10:816-820 (2004); Kumar et al., Nature 448:39-43 (2007); U.S. Pat. Nos.6,344,445, 6,924,144, 6,521,457). Furthermore, gene therapy approaches,for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 andDavidson, WO 04/013280, can be used to express nucleic acid molecules inthe brain.

Various non-viral delivery systems are known and can be used toadminister a therapeutic polynucleotide (e.g., siRNA) to the brain,e.g., encapsulation in liposomes, microparticles, microcapsules, byiontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for exampleGonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722) or by the use ofconjugates. For example, chemically modified liposomes encapsulatingsmall hairpin RNA can be conjugated to monoclonal antibodies specificfor specific endogenous receptors (e.g., such as insulin andtransferrin) located on the blood brain barrier and brain cellularmembranes (see Boado, Pharm. Res. 24(9):1772-1787 (2007)). As a furtherexample, siRNA duplexes can be delivered with the combined use ofreceptor specific antibody delivery systems and avidin-biotintechnology. The siRNA can be mono-biotinylated in parallel with theproduction of a conjugate of the targeting monoclonal antibody andstreptavidin (see Xia et al., Pharm Res. 24(12):2309-16 (2007)). Othermethods to deliver siRNA across plasma membranes in vivo includechemically modified siRNAs such as cholesterol-conjugated siRNAs (seeDiFiglia et al., Proc Natl Acad Sci USA. 104(43):17204-9 (2007); Wolfrumet al., Nature Biotech. 25(10):1149-1157 (2007); Soutschek et al.,Nature 432:173-178 (2003)).

C. Zinc Finger Proteins

Zinc finger proteins can be engineered or selected to bind to anydesired target site within a kinase gene of known sequence including,for example, PLK2. An exemplary motif characterizing one class of theseproteins (C₂H₂ class) is -Cys-(X)₂₋₄-Cys-(X)₁₂-His-(X)₃₋₅-His (where Xis any amino acid). A single finger domain is about 30 amino acids inlength, and several structural studies have demonstrated that itcontains an alpha helix containing the two invariant histidine residuesand two invariant cysteine residues in a beta turn co-ordinated throughzinc. In some methods, the target site is within a promoter or enhancer.In other methods, the target site is within the structural gene. In somemethods, the zinc finger protein is linked to a transcriptionalrepressor, such as the KRAB repression domain from the human KOX-1protein (Thiesen et al., New Biologist 2, 363-374 (1990); Margolin etal., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al.,Nucl. Acids Res. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad.Sci. USA 91, 4514-4518 (1994)). In some methods, the zinc finger proteinis linked to a transcriptional activator, such as VIP16. Zinc fingerproteins can also be used to activate expression of desired genes.Methods for selecting target sites suitable for targeting by zinc fingerproteins, and methods for designing zinc finger proteins to bind toselected target sites are described in WO 00/00388. Methods forselecting zinc finger proteins to bind to a target using phage displayare described by EP.95908614.1. The target site used for design of azinc finger protein is typically of the order of 9-19 nucleotides.

For example, proteins have been described that have the ability totranslocate desired nucleic acids across a cell membrane. Typically,such proteins have amphiphilic or hydrophobic subsequences that have theability to act as membrane-translocating carriers. For example,homeodomain proteins have the ability to translocate across cellmembranes. The shortest internalizable peptide of a homeodomain protein,Antennapedia, was found to be the third helix of the protein, from aminoacid position 43 to 58 (see, e.g., Prochiantz, Current Opinion inNeurobiology 6:629-634 (1996). Another subsequence, the h (hydrophobic)domain of signal peptides, was found to have similar cell membranetranslocation characteristics (see, e.g., Lin et al., J. Biol. Chem.270:14255-14258 (1995)). Such subsequences can be used to translocateoligonucleotides across a cell membrane. Oligonucleotides can beconveniently derivatized with such sequences. For example, a linker canbe used to link the oligonucleotides and the translocation sequence. Anysuitable linker can be used, e.g., a peptide linker or any othersuitable chemical linker.

D. Antibodies

Kinase activity can be reduced by administering anti-kinase (e.g.,anti-PLK2) antibodies, both intact and binding fragments thereof, suchas Fabs, Fvs, which specifically bind to a kinase of the invention.Usually the antibody is a monoclonal antibody although polyclonalantibodies can also be expressed recombinantly (see, e.g., U.S. Pat. No.6,555,310). Examples of antibodies that can be expressed include mouseantibodies, chimeric antibodies, humanized antibodies, veneeredantibodies and human antibodies. Chimeric antibodies are antibodieswhose light and heavy chain genes have been constructed, typically bygenetic engineering, from immunoglobulin gene segments belonging todifferent species (see, e.g., Boyce et al., Annals of Oncology14:520-535 (2003)). For example, the variable (V) segments of the genesfrom a mouse monoclonal antibody may be joined to human constant (C)segments. A typical chimeric antibody is thus a hybrid proteinconsisting of the V or antigen-binding domain from a mouse antibody andthe C or effector domain from a human antibody. Humanized antibodieshave variable region framework residues substantially from a humanantibody (termed an acceptor antibody) and complementarity determiningregions substantially from a mouse-antibody, (referred to as the donorimmunoglobulin). See Queen et al., Proc. Natl. Acad. Sci. USA86:10029-10033 (1989) and WO 90/07861, U.S. Pat. No. 5,693,762, U.S.Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,530,101 andWinter, U.S. Pat. No. 5,225,539. The constant region(s), if present, arealso substantially or entirely from a human immunoglobulin. Antibodiescan be obtained by conventional hybridoma approaches, phage display(see, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO92/01047), use of transgenic mice with human immune systems (Lonberg etal., WO93/12227 (1993)), among other sources. Nucleic acids encodingimmunoglobulin chains can be obtained from hybridomas or cell linesproducing antibodies, or based on immunoglobulin nucleic acid or aminoacid sequences in the published literature.

Antibodies can be administered by intravenous injection. A portion ofthe injected antibodies will cross the blood-brain barrier.Alternatively antibodies can be administered directly to the brain(e.g., by intraventricular or intrathecal injection). Antibodies may beinternalized by synuclein-expressing cells by endocytosis.Alternatively, antibodies may be linked to carrier moiety thatfacilitates transport across the cell membrane.

In one embodiment an intrabody is used to reduce PLK2 activity. The term“intrabody” or “intrabodies” refers to intracellularly expressedantibody constructs, usually single-chain Fv antibodies, directedagainst a target inside a cell. Nam, et al. (2002) Methods Mol. Biol.193:301; der Maurr et al. (2002) J. Biol Chem November 22;277(47):45075; Cohen (2002) Methods Mol Biol 178:367. The scFv gene canbe transferred into cells, where scFv protein expression can modulatethe properties of its target, e.g. PLK2, sometimes extinguishing proteinfunction and causing a phenotypic knockout. Indeed, the scFv intrabodycan be expressed in the cytoplasm and directed to any cellularcompartment where it can target intracellular proteins and elicitspecific biological effects. Intrabodies thus provide effective meansfor blocking or modulating the activity of proteins such as PLK2.

E. Kinase Inhibitors to Modulate Activity

In addition to the biological molecules discussed above, small moleculecompounds can be used to modulate (usually inhibit) expression oractivity of kinases. As discussed below, known kinase inhibitors can bescreened for desired target specificity and other properties, andadditional inhibitors can be identified based on target specificity.PLK2 or GRK6 are preferred kinases for inhibition because they arecandidates for directly phosphorylating alpha-synuclein. PLK2 is aparticularly preferred kinase because it has been shown to phosphorylatealpha-synuclein to a much higher level than GRK6 or other kinases testedherein.

Other kinase targets include APEG1, CDC7 μl, MET GRK1, GRK2, GRK6,IKBKB, CKII and GRK7 are also preferred kinases for inhibition becausethey are likely to be indirect activators of the direct kinase(s).PRKG1, MAPK13, and GAK are preferred kinases for activation in Lewy bodydiseases because they are negative regulators of the phosphorylation ofalpha-synuclein. Alternatively, these kinases themselves or fragments ormimetics thereof having similar activity can be used directly asinhibitors of alpha-synuclein phosphorylation. Synphilin can be used asan alternative therapeutic target for inhibition of alpha-synucleinphosphorylation. For example, synphilin can be added to an assay havingalpha-synuclein and PLK-2 expression and inhibitors of synphilinidentified.

Compounds to be screened for capacity to modulate expression or activityof kinases include the modulators of expression described in Section IV.These compounds also include many known examples of kinase inhibitors,some of which are already approved for therapeutic uses or in clinicaltrials, usually for treatment of cancer. Lead structures includequinazolines, pyrido[d]- and pyrimidol[d]pyrimidines,pyrazolo[d]-pyrimidienes, pyrrolo[d]pyrimidines, pheylamino-pyrimidines,1-oxo-3-aryl-1H-indene-2-carboxylic acid derivatives, and substitutedindolin-2-ones and natural products such as strauosporine (see Traxleret al., 2001, Medicinal Research Reviews 21:499-512). Some suchcompounds are commercially available from Calbiochem-Novabiochem Corp.(La Jolla, Calif.) including H89, Y27632, AT877 (fasudil hydrochloride),rottlerin, KN62, U0123, PD184352, PD98059, SB203580, SB202190,wortmannin, Li⁺, Ro 318220, chelerythrein and10-[3-(1-piperazinyl)propyl]-2-trifluoromethyl-phenothiazine (seeDavies, Biochem. J. 351, 95-105 (2000)). Other compounds currently inclinical trials include STI571 (Glivec™, a phenylamino-pyrimidinederivative) (Novartis), ZD1839 (Iressa) (AstraZeneca), OSI-774(Roche/OSI), PK1166 (Novartis), CI1033 (Pfizer/Wamer-Lambert), EKB-569(Wyeth-Ayerst), SU5416 (SUGEN), PTK787/ZK224584, aniline-phthalazinederivative (Novartis/Schering AG), SU6668 (Sugen), ZD6474 (AstraZeneca),and CEP2583 (Cephalon). Caveolin-1 is an example of a compound known tomodulate the activity of the GRK kinases. Examples of compounds known tomodulate the activity of the PLK2/SNK kinases include the RING-H2 domainof hVPS18 (human vacuolar protein sorting 18), and calcium- andintegrin-binding protein CIB.

In certain embodiments the therapeutic agent is a small molecule that isa thiazolidone, a quinazoline, a pyrimidine (e.g., apyrido[d]-pyrimidine, pyrimidol[d]pyrimidine, a pyrazolo[d]-pyrimidiene,a pyrrolo[d]pyrimidine, a pheylamino-pyrimidines, or aphenylamino-pyrimidine derivative); an indazole-pyridine derivative, acarboxylic acid derivative (e.g., a 1-oxo-3-aryl-1H-indene-2-carboxylicacid derivative), a substituted indolin-2-one, an aniline-phthalazinederivative; a quinolinone derivative, a benzthiazole-3 oxide compound,an azaindazole compound, or a dihydropteridinone. In certain embodimentsthe therapeutic agent is a small molecule protein kinase inhibitordescribed in US 20070203143; US 2007/0179177; US 2007/0135387; US2007/0010565; US 2007/0037862; US 2007/0010566; US 2006/0074119; US2006/0079503; US 2006/0223833; US 2005/0014761; US 2004/0176380; US2006/0040997; U.S. Pat. No. 6,861,422; US 2005/0014760; US 2006/0025411;US 2004/0176380; US 2005/0014761; US 2007/0203143; US 2007/0072833; US2007/0135387 or WO 03087095. Each of the aforelisted publications isincorporated herein by reference.

Compounds may be synthetic or can be obtained from natural sources, suchas, e.g., marine microorganisms, algae, plants, and fungi. Othercompounds that can be tested include compounds known to interact withalpha-synuclein, such as synphilin. Alternatively, compounds can be fromcombinatorial libraries of agents, including peptides or smallmolecules, or from existing repertories of chemical compoundssynthesized in industry, e.g., by the chemical, pharmaceutical,environmental, agricultural, marine, cosmeceutical, drug, andbiotechnological industries. Combinatorial libraries can be produced formany types of compounds that can be synthesized in a step-by-stepfashion. Such compounds include polypeptides, proteins, nucleic acids,beta-turn mimetics, polysaccharides, phospholipids, hormones,prostaglandins, steroids, aromatic compounds, heterocyclic compounds,benzodiazepines, oligomeric N-substituted glycines and oligocarbamates.Large combinatorial libraries of compounds can be constructed by theencoded synthetic libraries (ESL) method described in Affymax WO95/12608, Affymax WO 93/06121, Columbia University WO 94/08051,Pharmacopeia WO 95/35503 and Scripps WO 95/30642 (each of which isincorporated herein by reference in its entirety for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980. Compounds to be screened can also be obtainedfrom governmental or private sources, including, e.g., the NationalCancer Institute's (NCI) Natural Product Repository, Bethesda, Md., theNCl Open Synthetic Compound Collection, Bethesda, Md., NCl'sDevelopmental Therapeutics Program, or the like. Compounds can include,e.g., pharmaceuticals, therapeutics, environmental, agricultural, orindustrial agents, pollutants, cosmeceuticals, drugs, organic compounds,lipids, glucocorticoids, antibiotics, peptides, proteins, sugars,carbohydrates, and chimeric molecules.

As discussed above, kinase inhibitors that preferentially inhibit PLK2are of particular value. As is shown in Examples 13-16, below, weassayed the effect of several kinase inhibitors levels ofphosphorylation of alpha-synuclein in rat ventral mesencephalon andmouse cortical cell cultures, and other cells.

An exemplary compound for use according to the invention is the compoundBI2536 having the structure:

As demonstrated in the Examples below, BI 2536(4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pteridinyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide;also called ELN-481574-2;) reduced phosphorylation of alpha synuclein ina variety of cell types. BI 2536 inhibits PLK1, PLK2 and PLK3 (see,Steegmaier et al., 2007, Current Biology, 17:316-322) and does notinhibit PLK4 (Johnson et al., 2007, Biochemistry 46:9551-9563).Steegmaier et al. reported an IC₅₀ of 0.83 nM for PLK1, 3.5 nM for PLK2and 9 nM for PLK3. In tests described in Example 15, below, BI2536 wasshown to have 16-fold selectivity for PLK2 (IC₅₀ 11 nM) and 13-foldselectivity for PLK3 (IC₅₀ 14 nM). BI 2536 has category IV PLK2specificity (Inhibits PLK2 activity and has no, or reduced, effect onPLK4) and is a candidate for Parkinson's disease therapeutics.

Accordingly, in one aspect the invention provides a method forinhibiting phosphorylation of alpha-synuclein in a mammalian cell bycontacting the cell with an amount of BI 2536 that reduces PLK2 activityin the cell such that phosphorylation of alpha-synuclein is reduced. Ina related aspect, the invention provides a method of treating a patientdiagnosed with Parkinson's disease by administering a therapeuticallyeffective amount of BI 2536.

U.S. Pat. No. 6,861,422, incorporated herein by reference, describes BI2536 and structurally related dihydropteridinone kinase inhibitors.Dihydropteridinone compounds that inhibit PLK2 are useful for inhibitingphosphorylation of synuclein.

Another exemplary compound for use according to the invention is thecompound ELN-481080 having the structure:

See, McInnes et al., 2006, Current Topics in Medicinal Chemistry5:181-97 (Compound 8). Also see US 2006/0040997, “Benzthiazole-3 oxidesuseful for the treatment of proliferative disorders” incorporated hereinby reference. See Example 14, below.

Several inhibitors of polo-like kinases are described in Johnson et al.,2007, “Pharmacological and functional comparison of the polo-like kinasefamily: insight into inhibitor and substrate specificity” Biochemistry46:9551-63. Of those characterized in that study, several preferentiallyinhibit PLK2 and may be used as therapeutic agents.

For example, CHIR-258 (3) is a multitarget growth factor kinaseinhibitor developed for treatment of t(4; 14) multiple myeloma (Trudelet al., 2005, Blood. 105:2941-8).

K_(i) values for CHIR-258 for PLK1, PLK2, PLK3 and PLK4 are >20,0.85, >20, and 1.4 uM respectively meaning CHR-258 is a Type VIinhibitor (inhibits PLK2 activity and has reduced effect on PLK1, PLK3and PLK4). Although CHIR-258 is an inhibitor of receptor tyrosinekinases, warranting study of the side effect profile, targeted deliveryto the brain and/or particular treatment regimens can be investigated todetermine whether the compound has an acceptable therapeutic index fortreatment and prevention of Parkinson's Disease, and a clinicallyacceptable side effect profile. CHIR-258 and related quinolinonederivatives are described in WO03087095, incorporated herein byreference.

Sunitinib (SU11248) (4) is an example of a Type IV inhibitor, based onthe Johnson et al., 2007, studies (inhibits PLK2 activity with reducedeffect on PLK1 or PLK3). Sunitinib was identified as an inhibitor ofFLT3 receptor tyrosine kinase (RTK) approved for treatment of advancedrenal cell carcinoma and gastrointestinal stromal tumors that arerefractory or intolerant to imatinib (Gleevec). O'Farrell et al., 2003,Blood 101:3597-605.

5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}thiophene-2-carboxamide(5) is an example of a Type III agent (inhibits PLK2 activity and hasreduced effect on PLK4). The compound was initially identified as aselective thiophene benzimidazole ATP-competitive inhibitor of PLK1 andPLK3 for treatment of neoplasms. Lansing et al., 2007, Mol Cancer Ther.6:450-9. Also see US 20060074119, incorporated herein by reference,describing other inhibitors of PLKs.

Compounds 6 and 7 are indazole-pyridine based inhibitors of proteinkinase B/Akt proposed as antitumor agents (see Woods et al., 2006,Bioorg Med. Chem. 14:6832-46). Each has a lower K_(i) for PLK2 than forPLK1 or PLK3 (Type IV inhibitor) and may be used for treatment ofParkinson's disease.

In some embodiments the agent is a naturally occurring agent. In someembodiments the agent has a molecular weight less than 4000, sometimesless than 3000, sometimes less than 2000, usually less than 1000, andsometimes less than 500.

In certain embodiments the therapeutic agent is a small molecule otherthan a thiazolidone. In certain embodiments the therapeutic agent is asmall molecule other than X, where X is one of, or an independentlyselected one or more of, (i) a thiazolidone, (ii) a quinazoline, (iii) apyrimidine, (iv) pyrido[d]-pyrimidine, (v) a pyrimidol[d]pyrimidine,(vi) a pyrazolo[d]-pyrimidiene, (vii) a pyrrolo[d]pyrimidine, (viii) apheylamino-pyrimidines, (ix) phenylamino-pyrimidine derivative, (x) aindazole-pyridine derivative, (xi) a carboxylic acid derivative, (xii) a1-oxo-3-aryl-1H-indene-2-carboxylic acid derivative, (xiii) asubstituted indolin-2-one, (xiv) an aniline-phthalazine derivative; (xv)a quinolinone derivative, (xvi) a thiazolidinone compound, (xvii) abenzthiazole-3 oxide (xviii) a dihydropteridinone or (xix) anazaindazole compound.

In certain embodiments the therapeutic agent is a small molecule withthe proviso it is not a compound described in US 2007/0179177. Incertain embodiments the therapeutic agent is a small molecule with theproviso it is not a compound described in US 2007/0010565. In certainembodiments the therapeutic agent is a small molecule with the provisoit is not a compound described in US 2007/0037862. In certainembodiments the therapeutic agent is a small molecule with the provisoit is not a compound described in 2007/0010566. In certain embodimentsthe therapeutic agent is a small molecule with the proviso it is not acompound described in 2006/0079503. In certain embodiments thetherapeutic agent is a small molecule with the proviso it is not acompound described in US 2006/0223833. Each of the aforelistedpublications is incorporated herein by reference.

VI. Methods of Treatment

The invention provides several methods of preventing or treating LewyBody disease in patients suffering from or at risk of such disease.Therapeutic agents include any of the agents described above thatinhibit phosphorylation of alpha-synuclein and/or reduce total levels ofalpha-synuclein.

The experimental examples below provide strong evidence that PLK2 is asynuclein kinase. Thus, in a particular aspect, the invention provides amethod of effecting treatment or prophylaxis of an LB disease byadministering to a patient suffering from or at risk of the disease aneffective regime of an agent effective to suppress activity orexpression of PLK2. It is preferred that the agent shows a high level ofspecificity for PLK2.

Patients amenable to treatment include individuals at risk of disease ofa LBD but not showing symptoms, as well as patients presently showingsymptoms. Therefore, the present methods can be administeredprophylactically to individuals who have a known genetic risk of a LBD.Such individuals include those having relatives who have experiencedthis disease, and those whose risk is determined by analysis of geneticor biochemical markers. Genetic markers of risk toward PD includemutations in the alpha-synuclein or Parkin, UCHLI, LRRK2, and CYP2D6genes; particularly mutations at positions 30 and 53 of thealpha-synuclein gene. Another genetic marker of risk toward PD includesmeasuring the levels or SNCA dosage or transcription. Individualspresently suffering from Parkinson's disease can be recognized from itsclinical manifestations including resting tremor, muscular rigidity,bradykinesia and postural instability.

In some methods, the patient is free of clinical symptoms or riskfactors for any amyloidogenic disease other than one characterized byLewy bodies. In some methods, the patient is free of clinical symptomsor risk factors of any disease characterized by extracellular amyloiddeposits.

In some methods the patient is not diagnosed with cancer and/orAlzheimer's disease.

Treatment typically entails multiple dosages over a period of time.Treatment can be monitored by assaying signs or symptoms of the diseasebeing treated relative to base line measurements before initiatingtreatment. In some methods, administration of an agent results inreduction of intracellular levels of aggregated alpha-synuclein. In somemethods, administration of the agent results in a reduction in levels ofphosphorylated synuclein. In some methods, administration of an agentresults in improvement in a clinical symptom of a LBD, such as motor orcognitive function in the case of Parkinson's disease.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of aLBD in regime comprising an amount and frequency of administration ofthe composition or medicament sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingphysiological, biochemical, histologic and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. In therapeuticapplications, compositions or medicates are administered to a patientsuspected of, or already suffering from such a disease in a regimecomprising an amount and frequency of administration of the compositionsufficient to cure, or at least partially arrest, the symptoms of thedisease (physiological, biochemical, histologic and/or behavioral),including its complications and intermediate pathological phenotypes indevelopment of the disease. An amount adequate to accomplish therapeuticor prophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. A combination of amount and dosagefrequency adequate to accomplish therapeutic or prophylactic treatmentis defined as a therapeutically or prophylactically-effective regime.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. Guidance can be obtainedfrom the dosing schedules of kinase inhibitors currently approved or inclinical trials for other indications. Dosages in the range of 0.1-1000mg, preferably 10-500 mg, may be used. Frequency of dosing (e.g., daily,weekly or monthly) depends on the half-life of the drug. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patent can beadministered a prophylactic regime.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intrathecal, intraperitoneal, intranasal or intramuscular means forprophylactic and/or therapeutic treatment. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. In some methods, agentsare administered as a sustained release composition or device, such as aMedipad™ device. Small molecules that pass through the blood brainbarrier sufficiently are usually administered orally, but can also beadministered intravenously.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofLBD. Agents of the invention can also be administered in conjunctionwith other agents that increase passage of the agents of the inventionacross the blood-brain barrier.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose(TM), agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl. Compositions for parenteral administration aretypically substantially sterile, substantially isotonic and manufacturedunder GMP conditions of the FDA or similar body.

Compositions may be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above (see Langer, Science 249, 1527 (1990) andHanes, Advanced Drug Delivery Reviews 28, 97-119 (1997). The agents ofthis invention can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein. Alternatively, transdermal delivery canbe achieved using a skin patch or using transferosomes (Paul et al.,Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta1368, 201-15 (1998)).

VII. Lewy Body Diseases

Lewy Body Diseases (LBD) are characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al., Clinical andpathological diagnosis of dementia with Lewy bodies (DLB): Report of theCDLB International Workshop, Neurology (1996) 47:1113-24). Lewy Bodiesare spherical protein deposits found in nerve cells. Their presence inthe brain may disrupt the brain's normal function interrupting theaction of chemical messengers including acetylcholine and dopamine. LewyBody diseases include Parkinson's disease (including idiopathicParkinson's disease (PD)), Diffuse Lewy Body Disease (DLBD) also knownas Dementia with Lewy Bodies (DLB), Combined Alzheimer's and Parkinsondisease and multiple system atrophy (MSA). DLBD shares symptoms of bothAlzheimer's and Parkinson's disease. DLBD differs from Parkinson'sdisease mainly in the location of Lewy Bodies. In DLBD Lewy Bodies formmainly in the cortex. In Parkinson's disease, they form in selectedregions throughout the brain stem, midbrain, and in advanced disease,cerebral cortex. See, Braak et al., 2003, “Staging of brain pathologyrelated to sporadic Parkinson's disease” Neurobiology of Aging24:197-211. Other Lewy Body diseases include Pure Autonomic Failure,Lewy body dysphagia, Incidental LBD, Inherited LBD (e.g., mutations ofthe alpha-synuclein gene, PARK3 and PARK4) and Multiple System Atrophy(e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration andShy-Drager Syndrome).

VIII. Identification of Modulators of Kinases that Directly orIndirectly Phosphorylate Alpha-Synuclein

Agents that modulate expression or activity of a kinase that directly orindirectly phosphorylates alpha-synuclein can be identified by a varietyof assays. Particularly preferred are agents that inhibit kinases PLK2or GRK6, or APEG1, CDC7L1, MET GRK1, GRK2, GRK6, IKBKB, CKII and GRK7,or agents that activate PRKG1, MAPK13, and GAK. Agents that modulateexpression can be identified in cell-based assays in which an agentunder test is introduced into a cell expressing alpha-synuclein and akinase that directly or indirectly effects phosphorylation of thealpha-synuclein or modulates levels of total alpha-synuclein.Optionally, particularly for PLK2, synphilin can be expressed as well toaugment activity of the kinase. The agent can be introduced directly orin the form of a nucleic acid encoding the agent and capable ofexpressing the agent. The cell can naturally express the alpha-synucleinand kinase or one or both of these can be introduced into the cell bytransfection of a suitable nucleic acid. The effect of the agent onexpression of the kinase can be measured directly from the level of thekinase or its mRNA, or indirectly by measuring the level ofphosphorylated alpha-synuclein or total alpha-synuclein as describedabove. The level of kinase mRNA can be assayed by a hybridization typeassay. The level of the kinase can be assayed by an immunoassay.Optionally, the kinase is tagged with a peptide label such as Flag™(Hopp et al., BioTechnology 6, 1204-1210 (1988)) to facilitatedetection. An agent that decrease the level of the kinase, decrease thelevel of phosphorylation of alpha-synuclein and/or decrease the level oftotal alpha-synuclein relative to similar control cells not treated withan agent have a pharmacological activity potentially useful fortreatment of Lewy body diseases.

Agents are also screened for activity to modulate activity of a kinasesuspected of phosphorylating alpha-synuclein or increasing total levelsof alpha-synuclein. An initial screen can be performed to select asubset of agents capable of specifically binding to a kinase. Such anassay can be performed in vitro using an isolated kinase or fragmentthereof having kinase activity.

In one embodiment an agent that reduces alpha-synuclein phosphorylationin a mammalian cell expressing alpha-synuclein, and which may be used asa therapeutic agent, is identified based on the following criteria: a)reduces activity of PLK2 in a cell expressing PLK2; and b) does notreduce activity of PLK1 in a cell expressing PLK1, or reduces activityof PLK1 at a higher EC₅₀ than for PLK2; and/or c) does not reduceactivity of PLK3 in a cell expressing PLK3, or reduces activity of PLK3at a higher EC₅₀ than for PLK2; and/or d) does not reduce activity ofPLK4 in a cell expressing PLK4, or reduces activity of PLK4 at a higherEC₅₀ than for PLK2. In one embodiment an agent is selected that meetsall of criteria a-d. The mammalian cell may be a cell overexpressingalpha-synuclein (e.g., transfected with a vectors expressing exogenous,e.g., human, alpha-synuclein). The mammalian cell may be aneuronally-derived cell such as, for example, cells from mouse corticalcell cultures, rat ventral mesencephalon cell cultures, or otherneuronal cells from humans or non-human mammals.

Agents identified by such a screen can then be assayed functionally.Agents can also be directly assayed functionally without the bindingassay. For a kinase that directly phosphorylates alpha-synuclein,modulators can be screened by an in vitro assay combining the kinase,alpha-synuclein, ATP and the modulator in comparison with a control inwhich the modulator is omitted. Optionally, synphilin can be included aswell to increase phosphorylation particularly if the kinase is PLK2. Themodulator has potentially useful pharmacological activity if it reducesthe level of phosphorylation beyond the margin of typical experimentalerror relative to the control.

Agents can also be screened in cells expressing alpha-synuclein and thekinase under test, and optionally, particularly if the kinase is PLK2,synphilin. Such screens are effective regardless of whether the kinasephosphorylates alpha-synuclein directly or indirectly, or otherwiseaffects levels of alpha-synuclein. Cells are contacted with the agentand levels of total alpha-synuclein and phosphorylated alpha-synucleinare measured, as above, relative to a control cell not treated with theagent. A reduction in the level of phosphorylated alpha-synuclein ortotal alpha-synuclein relative to the corresponding level in a controlcell not treated with the agent, beyond the margin of typicalexperimental error, is an indication that the compound haspharmacological activity potential useful in treating Lewy bodydiseases. Agents may also be screened in cells for ability to reduceaggregation of alpha-synuclein in the cell.

Agents can also be screened in transgenic animal models of Lewy bodydisease, alone or in combination with the other assays described above.Total levels of alpha-synuclein, phosphorylated alpha-synuclein orLewy-like bodies or other indica of Lewy Body pathology or symptoms aremeasured in a transgenic animal treated with an agent under testrelative to corresponding levels in a similar control animal not treatedwith the agent. A reduction in one or more of these levels is anindication, the agent has pharmacological activity potentially useful intreating Lewy body diseases.

The kinase used in the above assays and cellular and transgenic modelsis preferably a human kinase having a sequence in one of the referencesor accession numbers provided in this disclosure. However, allelic(variants within a species) and species variants (variants betweenspecies) of such a kinase can also be used, as can variants having atleast 90% sequence identity to such a kinase. For subsequent clinicaluse, agents identified by such assays are capable of modulating theactivity or expression of a natural kinase, preferably a form occurringin humans.

IX. Transgenic Animal and Cellular Models of Lewy Body Disease

Transgenic animal models are useful for testing the capacity of kinasesto effect phosphorylation of alpha-synuclein and formation of Lewy-likebodies as described above. Transgenic animals are also useful forscreening agents for activity in modulating phosphorylation orproduction of alpha-synuclein. Particularly preferred are agents thatinhibit or are suspected of inhibiting kinases including PLK2 and GRK6,or APEG1, CDC7L1, MET GRK1, GRK2, GRK6, IKBKB, CKII and GRK7. Alsopreferred are agents that activate or are suspected of activatingkinases PRKG1, MAPK13, and GAK. Further, knockout animals (i.e., animalsin which an endogenous kinase is inactivated either by insertionalinactivation or trans inhibition by an siRNA, zinc finger protein or thelike) are useful for identifying the effect of eliminating activity of akinase on an animal. For example, analysis of a PLK2-knockout mouse canindicate whether inhibitors of PLK2 have any side effects. Analogousknockouts can reveal similar information for other kinases.

In general, transgenic models have a genome comprising analpha-synuclein transgene in operable linkage with one or moreregulatory sequences to ensure its expression. Expression of thetransgene leads to Lewy-body like deposits of alpha-synuclein in thebrain of the animal. Several such transgenic animals have been describedin the scientific and patent literature (see Masliah et al., Am. J.Pathol. (1996) 148:201-10 and Feany et al., Nature (2000) 404:394-8)),U.S. Pat. No. 5,811,633 (for transgenic animals with a mutant form ofAPP). Some transgenic animals express variant or mutant alpha-synuclein,such as familial mutants A30P, A53T, and E46K of alpha synuclein. Sometransgenic animals have an additional transgene, such as a transgeneencoding a kinase as described above. Transgenic animals bearing atransgene expressing alpha-synuclein protein can also be crossed withother transgenic models of neurogenic disease, such as models ofAlzheimer's disease. For example, transgenic animals bearing a transgeneexpressing a truncated alpha-synuclein protein can be crossed withtransgenic animals bearing a transgene expressed APP with a FAD mutationas described by e.g., Games et al., Nature 373, 523 (1995) McConlogue etal., U.S. Pat. No. 5,612,486, Hsiao et al., Science 274, 99 (1996);Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292(1997); Borchelt et al., Neuron 19, 939-945 (1997)). The procedure forperforming such a cross is described by e.g., Masliah et al., PNAS USA98:12245-12250 (2001), which reports a cross between transgenic miceexpressing a full length alpha-synuclein with PDAPP mice as described byGames et al Transgenic animals of the invention are preferably rodents,such as mice or rats, or insects, such as Drosophila. Transgenic animalscan be produced by introduction of a transgene at the germline stage inwhich case all or substantially all (except for rare loss throughsomatic mutation) of the cells of the transgenic animal include thetransgene integrated into the genome. Transgenes can be introduced bymicroinjection, nuclear transfer or viral infection into cells oranimals. Adeno Associated Viruses and Lentiviruses are particularlysuitable for the latter. Alternatively, transgenes can be introduced byviral infection into the brain of the animal. Such transgenes are notpart of the germline of recipient animals but can be targeted to regionsof the brain responsible for disease (e.g., the substantia nigra). Suchanimal models incorporate an alpha-synuclein into the genome of braincells and are disposed to develop at least one characteristic of LBDdisease. Lentiviruses provide a suitable vehicle for so introducing analpha-synuclein transgene into the brain (see Brain Pathology 13,364-372 (2003); Bjorklund, Trends Neurosci. 26, 386-92 (2003), Lothariuset al., J. Biol. Chem. 277, 38884-94 (2002), Zhou et al., Brain Research866, 33-43 (2000)). Transgenic animals can also include a transgenecapable of expressing one of the kinases of the invention (e.g., anucleic acid encoding the kinase in operable linkage with regulatoryelements to ensure its expression in the brain of an animal), instead ofor as well as a transgene expressing alpha synuclein. Optionally, atransgene expressing synphilin can be included as well. Some cellularmodels express variant or mutant alpha-synuclein, such as familialmutants A30P, A53T, and E46K of alpha synuclein. Some cells have anadditional transgene, such as a transgene encoding a kinase as describedabove, e.g., PLK2.

Cellular models of Lewy body disease can also be used in the screeningmethods of the invention. Cells transfected with alpha-synuclein forminclusion bodies containing aggregated alpha-synuclein. The transformedcells are preferably neuronal cells, such as GT1-7 neuronal cells (Hsueet al. Am. J. Pathol. 157:401-410 (2000)), PC12 cells or SY5Yneuroblastoma cells. PEAK and/or HCC cells can also be used (see Example10). The cells are preferably human cells. A vector comprising a segmentencoding a form of alpha-synuclein operably linked to one or moreregulatory sequences that ensure expression of the expression istransfected into the cells. Cells can also be transfected with a nucleicacid encoding a kinase of the invention as described above. Transfectedcells can be used to screen agents for activity in clearingalpha-synuclein inclusions. An exemplary cellular model is identified inExample 10 in which HCC neuronal cells are transfected with synucleinand PLK2 with the result that aggregation and phosphorylation of thesynuclein matching LB formation occurs. In order to identify inhibitorsof the kinase, the cell is contacted with the inhibitor and a reductionin the amount of phosphorylation and/or aggregation is identified.

X. Alpha-Synuclein Isolation and Phosphorylation

Human alpha-synuclein is a polypeptide of 140 amino acids having thefollowing amino acid sequence:

(SEQ ID NO:1) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVHGVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQEGILEDMPVDP DNEAYEMPSE EGYQDYEPEA

(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6); GenBankaccession number: P37840). The protein has three recognized domains, aKTKE repeat domain covering amino acids 1-61, a NAC (Non-AmyloidComponent) domain running from about amino acids 60-95, and a C-terminalacidic domain running from about amino acid 98 to 140.

Unless otherwise apparent from the context, reference to alpha-synucleinincludes the natural human amino acid sequence indicated above as wellas analogs including allelic, species and induced variants (e.g., E83Q,A90V, A76T) having at least 90% sequence identity to natural humanalpha-synuclein. Amino acids of analogs are assigned the same numbers ascorresponding amino acids in the natural human sequence when the analogand human sequence are maximally aligned. Analogs typically differ fromnaturally occurring peptides at one, two or a few positions, often byvirtue of conservative substitutions. Some natural allelic variants aregenetically associated with hereditary LBD. The term “allelic variant”is used to refer to variations between genes of different individuals inthe same species and corresponding variations in proteins encoded by thegenes. Allelic variants include familial mutants or variants, such asE46K, A30P and A53T (the first letter indicates the amino acid in SEQ IDNO:1, the number is the residue position in SEQ ID NO:1, and the secondletter is the amino acid in the allelic variant). Analogs can includeany combination of allelic variants. The A53T variation is associatedwith enhanced levels of phosphorylation at position 129 ofalpha-synuclein in an individual having the mutation relative to thenorm of phosphorylation in undiseased individuals who lack the mutation.

Alpha-synuclein, its fragments, and analogs can be synthesized by solidphase peptide synthesis or recombinant expression, or can be obtainedfrom natural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif. Recombinant expression can be in bacteria, such as E. coli,yeast, insect cells or mammalian cells. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989).

A method was developed herein to prepare large amounts of wild-typephospho-S129 alpha-synuclein, mutant and/or familial forms in abacterial expression system. When recombinant hPLK2 was co-expressedwith alpha-synuclein in bacteria, the phospho-S129 alpha-synuclein thatwas produced in the cell was recovered with a very high yield andpurity. This is because, unlike most E. coli proteins, thealpha-synuclein could resist heating. After boiling of the bacteriallysate, alpha-synuclein purity reached about 95% before chromatography.

To co-express recombinant protein in a bacterial system, the plasmidsharboring each gene of interest were chosen be compatible within thebacterial cell by ensuring that they possessed a different origin ofreplication and a different antibiotic selection. The alpha-synucleingene was sub-cloned into a pDEST24 compatible vector, pCDF1b. BL21-DE3bacteria were then co-transformed with both the pDEST24 containingwild-type hPLK2 or hPLK2 constitutive mutant constructs without a GSTtag and the pCDF1b/AS plasmid. The bacterial lysates were boiled and thesupernatant, expected to contain alpha-synuclein was analyzed by Westernblot with an anti-phospho-S129-alpha-synuclein antibody (11A5), by usingthe SYPRO Ruby™ and ProZDiamond™ dyes (total protein and phospho-Ser/Thrspecific dye respectively), by SDS-PAGE and by mass spectrometry, withthe results that a fairly pure phospho-S129-alpha-synuclein product wasisolated that, upon analysis by mass spectrometry was revealed to bemore than 95% phosphorylated. To ensure that the final product was 100%phosphorylated and highly pure, the supernatant of the lastcentrifugation was passed through an 11A5-sepharose-affinitypurification column one or more times. Any contamination was removedusing HPLC.

XI. Examples Example 1 Screen for Kinases that Modulate Alpha-SynucleinPhosphorylation

To identify the kinase or kinases that phosphorylates α-synuclein atserine-129 an siRNA kinase library (Ambion) was screened on cellscontaining a quantifiable amount of phosphorylated α-synuclein. Humanembryonic kidney cell line HEK293 cells (PEAK cells) stably transfectedwith human wild-type α-synuclein under control of a CMV promoter(PEAK-Syn cells) were transfected with 100 nM siRNAs targeting 597 humankinases and were assayed by ELISA assays to quantitate total andphospho-synuclein levels. Ninety-five kinases with siRNAs that alteredthe percentage of phosphorylated alpha-synuclein were identified (seeTables 1-2). Of those, 28 belonged to the class of kinases thatphosphorylate serine residues and hence were capable of directlyphosphorylating α-synuclein at serine-129. Others were tyrosine kinases.Although tyrosine kinases do not phosphorylate α-synuclein at ser-129directly, they can act as upstream regulators of the alpha-synucleinkinase. Two of these ser/thr kinases, casein kinase 2 andcalcium/calmodulin dependent protein kinase II, have been reported tophosphorylate α-synuclein in vitro (Pronin et al, J. Biol. Chem. 275:26515-26522 (2000), Okochoa et al, J. Biol. Chem. 275: 390-397 (2000);Nakajo et al, Eur. J. Biochem. 217: 1057-1063 (1993) and a casein kinase2 inhibitor has been reported to decrease phospho-synuclein levels incells (Okochoa et al, 2000). Several of the GRK family members (althoughnot GRK6) have been reported to phosphorylate alpha-synuclein in vitro(Pronin et al, 2000). GRK2 expression in flies has been reported toincrease phospho-synuclein levels (Chen and Feany, Nature Neurosci. 8:657-663 (2005)).

In addition to kinases that lower phospho-synuclein levels, 99 kinaseswhose siRNAs altered total α-synuclein levels in the PEAK-Syn cells wereidentified in Table 2 and included fucokinase (FUK), Genbank numberNM_(—)145059; Protein Kinase N1 (PRKCL1, PKN1), Genbank numberNM_(—)002741; Doublecortin and CaM kinase-like 1 (DCAMKL1) NM_(—)004734;Branched chain Ketoacid dehydrogenase kinase (BCKDK) NM_(—)005881;Aurora Kinase C (AURKC, STK13); NM_(—)003160, Kinase suppressor of ras 2(FLJ25965), NM_(—)173598; FLJ32704; MAP2K6; and Tousled-like kinase 2(TLK2) NM_(—)006842. The mechanism of action may involve eitherregulation of alpha-synuclein turnover or synthesis (See Table 2).

Tables 1A, 1B and 1C show kinases whose inhibition modulates thephosphorylation at position ser-129. Table 1A, B, and C differ in thetype of kinase. Table 1A shows kinases that can phosphorylate serineresidues and often tyrosine and/or threonine as well. Table 1B showstyrosine kinases that cannot (so far as is known) modify serineresidues. Table 1C shows kinases not known to have phosphorylateproteins. Kinases from the upper portion of Table 1A are candidates fordirect phosphorylation of ser-129 of alpha-synuclein. Kinases from theupper part of Table 1B are also useful therapeutic targets via rolesindirectly phosphorylating alpha-synuclein. Proteins in the upper partof Table 1C are also useful therapeutic targets for the same reason.Cols. 1, 2 and 3 of each table indicate the gene name, kinase name andGenbank accession number of kinases. The next column indicates whetherinhibiting expression of the kinase decreased (“down”) or increased(“up”) phosphorylation. The next three columns indicate the number ofstandard deviations the measured level of phosphorylation departs fromthe mean in three independent experiments. The final two columnsindicate the kinase family (i.e., amino acid specificity) and group.

Tables 2 and 3 show kinases whose inhibition modulates the overalllevels of human alpha-synuclein without changing the percentage ofphosphorylation. Table 2 shows all of the kinases with the strongestreduction in levels of human alpha-synuclein. The columns are labeledsimilarly to Tables 1A, 1B and 1C.

Tables 1A-C: Complete list of Kinase candidates that reducephosphorylation

TABLE 1A Serine/Threonine Kinases Up/ # of # of # of Kinase Gene NameKinase Name Genbank Down SD SD SD Kinase Family Group GPRK6 Gprotein-coupled receptor NM_002082 Down 1.25 1.25 Ser/Thr AGC kinase 6PDPK1 3-Phosphoinositide NM_002613 Down 2.25 2.75 Ser/Thr AGC dependentprotein kinase-1 FLJ11159 RIO kinase 2 (yeast) NM_018343 Down 1.5 1.5Ser/Thr Atypical *APEG1 Aortic preferentially NM_005876 Down 1.75 2 2.25Ser/Thr/Tyr CAMK expressed gene 1 ARK5 AMP-activated protein NM_014840Down 1 1 1.5 Ser/Thr CAMK kinase family member 5 *CAMK1Calcium/calmodulin- NM_003656 Down 1.5 1.75 2 Ser/Thr CAMK dependentprotein kinase I SSTK Serine/threonine protein NM_032037 Down 1 1Ser/Thr CAMK kinase SSTK PHKG2 Phosphorylase kinase, NM_000294 Down 1 2Ser/Thr CAMK gamma 2 (testis) CASK Calcium/calmodulin- NM_003688 Down1.25 1.25 Ser/Thr CAMK dependent serine protein kinase PRKAA2 Proteinkinase, AMP- NM_006252 Down 1 1.75 Ser/Thr CAMK activated, alpha 2catalytic subunit CDK8 Cyclin-dependent kinase 8 NM_001260 Down 1 1 1.25Ser/Thr CMGC *CDC2L5 Cell division cycle 2-like 5 NM_003718 Down 1.251.5 Ser/Thr/Tyr CMGC ERK8 Extracellular signal- NM_139021 Down 1.5 1.5Ser/Thr CMGC regulated kinase 8 *CDK4 Cyclin-dependent kinase 4NM_000075 Down 1 1 Ser/Thr** CMGC CLK3 CDC-like kinase 3 NM_003992 Down1 1.5 Ser/Thr/Tyr CMGC PRP4 Pre-mRNA processing NM_003913 Down 1.25 1.75Ser/Thr GO factor 4 homolog B (yeast) CKIIA2 Casein kinase 2, alphaNM_001896 Down 1 1.5 Ser/Thr Other prime subunit PLKII/SNK Polo likekinase 2 NM_006622 Down 1 1.5 2.25 Ser/Thr Other CKIIA1 Casein kinase 2,alpha NM_001895 Down 1 1.75 2.25 Ser/Thr Other subunit MAP2K1mitogen-activated protein NM_002755 Down 1 1.75 Ser/Thr/Tyr STE kinasekinase 1 (MEK1; MKK1) MAP2K4 mitogen-activated protein NM_003010 Down 12 Ser/Thr/Tyr STE kinase kinase 4 (MEK4; MKK4; JNKK) MAP2K5mitogen-activated protein NM_002757 Down 1 1.25 Ser/Thr/Tyr STE kinasekinase 5 (MEK5; MKK5) TESK2 testis-specific kinase 2 NM_007170 Down 1 11.5 Ser/Thr/Tyr TKL RIPK3 receptor-interacting serine- NM_006871 Down 11.25 Ser/Thr/Tyr TKL threonine kinase 3 PRKG2 protein kinase, cGMP-NM_006259 Down 2.25 Ser/Thr AGC dependent, type II JIK TAO Kinase 3(MAP3K18) NM_016281 Down 2 Ser/Thr STE PAK6 p21(CDKN1A)-activatedNM_020168 Down 2.5 Ser/Thr STE kinase 6 *CAMK2D Calcium/calmodulin-NM_001221 Down 1.25 1.5 2 Ser/Thr CAMK dependent protein kinase II-delta *CDC7L1 CDC7 cell division cycle 7- NM_003503 Down 1.25 2 3Ser/Thr Other like 1 CDK5 Cyclin-dependent kinase 5 NM_004935 Up 1 11.25 Ser/Thr CMGC PRKWNK1 Protein kinase, lysine NM_018979 Up 1 1.5 6.25Ser/Thr Other deficient 1 CDC42BPB CDC42 binding protein NM_006035 Up2.5 3.75 Ser/Thr AGC kinase beta (DMPK-like) PRKCI protein kinase C,iota NM_002740 Up 1 1.75 Ser/Thr AGC PRKG1 Protein kinase, cGMP-NM_006258 Up 1 1.25 Ser/Thr AGC dependent, regulatory, Type I SMG1PI3-kinase-related kinase NM_015092 Up 1.25 1.5 Ser/Thr Atypical SMG1*BRD3 Bromodomain-containing NM_007371 Up 1.25 1.75 Ser/Thr Atypicalprotein 3 DAPK1 Death-associated protein NM_004938 Up 1 1.25 1.25Ser/Thr CAMK kinase 1 PASK PAS domain containing NM_015148 Up 1 1Ser/Thr CAMK serine/threonine kinase LOC283629 Chromosome 14 openNM_174944 Up 1 1.25 Ser/Thr/Tyr CAMK reading frame 20; Testis- specificserine kinase 4 CDC2 Cell division cycle 2, G1 to NM_001786 Up 1.25 2.75Ser/Thr/Tyr CMGC S and G2 to M MAPK13 Mitogen-activated proteinNM_002754 Up 1 1.25 1.75 Ser/Thr/Tyr CMGC kinase 13 STK35Serine/threonine kinase 35, NM_080836 Up 1 1.25 1.5 Ser/Thr Other Clik1GAK Cyclin G associated kinase NM_005255 Up 1 1.25 1.75 Ser/Thr OtherANKRD3 ankyrin repeat domain 3 NM_020639 Up 1 1.75 Ser/Thr/Tyr TKL IRAK3Interleukin-1 receptor- NM_007199 Up 1.5 1.75 Ser/Thr TKL associatedkinase 3 LIMK2 LIM domain kinase 2 NM_005569 Up 1 2 Ser/Thr/Tyr TKLPKMYT1 Protein kinase, membrane- NM_004203 Both 1.25 1.5 1.75Ser/Thr/Tyr Other associated, tyrosine/threonine 1 ADRBK2 adrenergic,beta, receptor NM_005160 Up 2 Ser/Thr AGC kinase 2 (GRK3; BARK2) AKT3v-akt murine thymoma viral NM_005465 Up 2 Ser/Thr AGC oncogene homolog 3(protein kinase B, gamma) CDK10 cyclin-dependent kinase NM_003674 Up5.75 Ser/Thr/Tyr CMGC (CDC2-like) 10 EIF2AK3 eukaryotic translationNM_004836 Up 2.5 Ser/Thr Other initiation factor 2-alpha kinase 3 BIKEBMP2 inducible kinase NM_017593 Up 6 Ser/Thr Other (BMP2K), transcriptvariant IKBKE inhibitor of kappa light NM_014002 Up 2 Ser/Thr Otherpolypeptide gene enhancer in B-cells, kinase epsilon SDCCAG43serologically defined colon NM_006648 Up 4.25 Ser/Thr/Tyr Other cancerantigen 43 FLJ10074 SCY1-like 2 (S. cerevisiae) NM_017988 Up 2.25Ser/Thr Other FLJ32685 hypothetical protein NM_152534 Up 3 Ser/Thr/TyrOther FLJ32685 NEK11 NIMA (never in mitosis NM_024800 Up 4 Ser/Thr/TyrOther gene a)-related kinase 11 TTK TTK protein kinase NM_003318 Up 2.5Ser/Thr/Tyr Other

TABLE 1B Tyrosine Kinases Up/ # of # of # of Kinase Kinase Gene NameKinase Name Genbank Down SD SD SD Family Group PDGFRA Platelet-derivedgrowth NM_006206 Down 1.5 2 Tyr TK factor receptor, alpha SRMSsrc-related kinase lacking C- NM_080823 Down 1.25 1.75 Tyr TK terminalregulatory tyrosine and N-terminal myristylation sites PTK6 Proteintyrosine kinase 6 NM_005975 Down 1.25 1.5 Tyr TK ZAP70 zeta-chain (TCR)associated NM_001079 Down 1.5 2 Tyr TK protein kinase 70 kDa ERBB4v-erb-b2 erythroblastic NM_005235 Down 1.25 1.5 Tyr TK leukemia viraloncogene homolog 4 (avian) IGF1R insulin-like growth factor 1 NM_000875Down 1 1.5 Tyr TK receptor MET met proto-oncogene NM_000245 Down 1 1.5Tyr TK (hepatocyte growth factor receptor) MERTK c-mer proto-oncogeneNM_006343 Down 1 1.25 Tyr TK tyrosine kinase JAK2 Janus kinase 2 (aprotein NM_004972 Down 1 1.5 Tyr TK tyrosine kinase) YES1 v-yes-1Yamaguchi sarcoma NM_005433 Up 1.25 1.25 Tyr TK viral oncogene homolog 1ERBB3 v-erb-b2 erythroblastic NM_001982 Up 1 1 Tyr TK leukemia viraloncogene homolog 3 (avian) EPHA7 EphA7 NM_004440 Up 1 1.25 1.5 Tyr TKBTK Bruton NM_000061 Up 1.25 1.5 Tyr TK agammaglobulinemia tyrosinekinase EPHB3 EphB3 NM_004443 Up 1 1.75 Tyr TK RET ret proto-oncogene(multiple NM_020975 Up 3 Tyr TK endocrine neoplasia and medullarythyroid carcinoma 1, Hirschsprung disease)

TABLE 1C No Protein Phosphorylation Activity Up/ # of # of # of KinaseKinase Gene Name Kinase Name Genbank Down SD SD SD Family Group C8FWTribbles homolog 1 NM_025195 Down 1 1 X CAMK CHK Choline kinaseNM_001277 Down 1 1.5 X GO FLJ13052 NAD kinase NM_023018 Down 1.25 1.75 XGO FLJ22055 Phosphatidylinositol-4- NM_024779 Down 1 1 X GO phosphate5-kinase, type II, gamma CKMT2 Creatine kinase, NM_001825 Down 1.75 2 XGO mitochondrial 2 (sarcomeric) DKFZP586B1621 DKFZP586B1621 protein,NM_015533 Down 1 1.25 X GO function unknown GK Glycerol kinase NM_000167Down 1.5 1.75 X GO ITPKC Inositol 1,4,5-trisphosphate NM_025194 Down 11.25 X GO 3-kinase C NME4 Non-metastatic cells 4, NM_005009 Down 1 1.25X GO protein expressed in NM23-H6 Non-metastatic cells 6, NM_005793 Down1.25 1.25 1.75 X GO protein expressed in (nucleoside-diphosphate kinase)RBSK Ribokinase NM_022128 Down 2.25 X GO ITPKC Inositol1,4,5-trisphosphate NM_025194 Down 1 1.25 X GO 3-kinase C PMVKPhosphomevalonate NM_006556 Both 1 1.25 2.5 X GO kinase GS3955 Tribbleshomolog 2 NM_021643 Up 1.25 1.25 X CAMK DGKI diacylglycerol kinase, iotaNM_004717 Up 3 X GO HK2 hexokinase 2 NM_000189 Up 2.25 X GO DGKGdiacylglycerol kinase, NM_001346 Up 2.25 X GO gamma 90 kDa NBP CoenzymeA synthase NM_025233 Up 2.75 X GO (COASY), DGKA Diacylglycerol kinase,NM_001345 Up 1.5 1.75 X GO alpha 80 kDa XYLB Xylulokinase homolog (H.influenzae) NM_005108 Up 1.25 3.5 X GO SPHK2 Sphingosine kinase 2NM_020126 Up 1.5 2 X GO PRKRA Protein kinase, interferon- NM_003690 Up 11 2.5 X GO inducible double stranded RNA dependent activator PIP5K2APhosphatidylinositol-4- NM_005028 Up 1 1 X GO phosphate 5-kinase, typeII, alpha

TABLE 2 Kinase whose inhibition modulates synuclein levels Gene TF Up/ #of # of Kinase Kinase Name Kinase Name Genbank Plates Down # of SD SD SDFamily Group PRKCD protein kinase C, NM_006254 1, 4, 7 Down 1 1.5Ser/Thr AGC delta GPRK2L G protein-coupled NM_005307 3, 6, 9 Down 1.251.5 Ser/Thr AGC receptor kinase 2-like (Drosophila) GPRK5 Gprotein-coupled NM_005308 3, 6, 9 Down 1.5 1.5 1.75 Ser/Thr AGC receptorkinase 5 AD034 RIO kinase 1 (yeast) NM_031480 10, 13, 16 Down 1.25 1.5Ser/Thr Atypical BRDT bromodomain, testis- NM_001726 12, 15, 18 Down 11.25 Ser/Thr Atypical specific EEF2K eukaryotic elongation NM_013302 12,15, 18 Down 1.5 2.25 Ser/Thr Atypical factor-2 kinase FASTKFas-activated NM_006712 12, 15, 18 Down 1.25 1.5 Ser/Thr Atypicalserine/threonine kinase LOC283629 Testis-specific serine NM_174944 21,24, 27 Down 1 1 Ser/Thr/ CAMK kinase 4 (TSSK4) Tyr STK22Dserine/threonine NM_032028 21, 24, 27 Down 1.25 1.25 Ser/Thr CAMK kinase22D (spermiogenesis associated); TSSK1 ALS2CR7 amyotrophic lateralNM_139158 28, 31, 34 Down 1 1.5 Ser/Thr CMGC sclerosis 2 (juvenile)chromosome region, candidate 7 CLK4 CDC-like kinase 4 NM_020666 30, 33,36 Down 1 1.5 Ser/Thr/ CMGC Tyr CDK5 cyclin-dependent NM_004935 30, 33,36 Down 1 2 Ser/Thr CMGC kinase 5 CSNK2A2 casein kinase 2, NM_001896 55,58, 61 Down 1 1.25 1.5 Ser/Thr Other alpha prime polypeptide MAP2K4mitogen-activated NM_003010 65, 68, 71 Down 1 2 Ser/Thr/ STE proteinkinase kinase 4 Tyr MAP2K1 mitogen-activated NM_002755 65, 68, 71 Down 11.75 Ser/Thr/ STE protein kinase kinase 1 Tyr MAP2K5 mitogen-activatedNM_002757 66, 69, 72 Down 1 1.25 Ser/Thr/ STE protein kinase kinase 5Tyr ANKRD3 ankyrin repeat NM_020639 83, 86, 89 Down 1.25 1.75 Ser/ThrTKL domain 3 (RIPK4) IRAK3 interleukin-1 NM_007199 83, 86, 89 Down 11.25 Ser/Thr TKL receptor-associated kinase 3 BMPR2 bone morphogeneticNM_001204 84, 87, 90 Down 1 2 Ser/Thr TKL protein receptor, type II(serine/threonine kinase) PRKG2 protein kinase, NM_006259 1, 4, 7 Down 2Ser/Thr AGC cGMP-dependent, type II CHEK2 CHK2 checkpoint NM_007194 19,22, 25 Down 3.25 Ser/Thr/ AGC homolog (S. pombe) Tyr CDK9cyclin-dependent NM_001261 28, 31, 34 Down 2 Ser/Thr CMGC kinase 9(CDC2- related kinase) CDK2 cyclin-dependent NM_001798 29, 32, 35 Down 2Ser/Thr CMGC kinase 2 CDKL3 cyclin-dependent NM_016508 29, 32, 35 Down2.25 Ser/Thr CMGC kinase-like 3 CDK10 cyclin-dependent NM_003674 29, 32,35 Down 3 Ser/Thr/ CMGC kinase (CDC2-like) Tyr 10 CDK7 cyclin-dependentNM_001799 30, 33, 36 Down 2 Ser/Thr CMGC kinase 7 (MO15 homolog, Xenopuslaevis, cdk-activating kinase) PK428 CDC42 binding NM_003607 40, 46, 52Down 2 Ser/Thr/ GO protein kinase alpha Tyr (DMPK-like) FLJ32685hypothetical protein NM_152534 57, 60, 63 Down 2.75 Ser/Thr/ OtherFLJ32685 Tyr NEK11 NIMA (never in NM_024800 57, 60, 63 Down 3.5 Ser/Thr/Other mitosis gene a)- Tyr related kinase 11 JIK TAO Kinase 3 NM_01628164, 67, 70 Down 2 Ser/Thr STE (MAP3K18) PAK6 p21(CDKN1A)- NM_020168 65,68, 71 Down 2.25 Ser/Thr STE activated kinase 6 KSR kinase suppressor ofNM_013571 83, 86, 89 Down 2.25 Ser/Thr/ TKL ras Tyr AMHR2 anti-MullerianNM_020547 83, 86, 89 Down 2 Ser/Thr/ TKL hormone receptor, Tyr type IILIMK2 LIM domain kinase 2 NM_005569 84, 87, 90 Down 2 Ser/Thr/ TKL TyrBCR breakpoint cluster NM_004327 11, 14, 17 Both 1 1.25 1.5 Ser/ThrAtypical region ROCK2 Rho-associated, NM_004850 2, 5, 8 Up 1 1 Ser/Thr/AGC coiled-coil containing Tyr protein kinase 2 SGK2serum/glucocorticoid NM_170693 2, 5, 8 Up 1.25 1.5 Ser/Thr AGC regulatedkinase 2 SGKL serum/glucocorticoid NM_013257 2, 5, 8 Up 1.25 1.25Ser/Thr/ AGC regulated kinase-like Tyr pknbeta protein kinase N3NM_013355 3, 6, 9 Up 1 1.75 Ser/Thr AGC PRKCH protein kinase C, etaNM_006255 3, 6, 9 Up 1 1.75 Ser/Thr AGC ROS1 v-ros UR2 sarcoma NM_00294410, 13, 16 Up 1 1.5 Ser/Thr/ TK virus oncogene Tyr homolog 1 (avian)CAMK1 calcium/calmodulin- NM_003656 20, 23, 26 Up 1.25 2 Ser/Thr CAMKdependent protein kinase I CAMK2B calcium/calmodulin- NM_172078 20, 23,26 Up 1.5 1.75 Ser/Thr/ CAMK dependent protein Tyr kinase (CaM kinase)II beta CAMK2D calcium/calmodulin- NM_001221 21, 24, 27 Up 1.5 2.25Ser/Thr CAMK dependent protein kinase (CaM kinase) II delta STK22Bserine/threonine NM_053006 21, 24, 27 Up 1.25 1.25 Ser/Thr/ CAMK kinase22B Tyr (spermiogenesis associated) STK29 serine/threonine NM_003957 21,24, 27 Up 1 2 Ser/Thr/ CAMK kinase 29 Tyr DYRK1B dual-specificityNM_004714 28, 31, 34 Up 1 1.25 Ser/Thr/ CMGC tyrosine-(Y)- Tyrphosphorylation regulated kinase 1B PCTK1 PCTAIRE protein NM_033018 28,31, 34 Up 1 1.25 Ser/Thr CMGC kinase 1 SRPK2 SFRS protein kinase 2NM_182692 30, 33, 36 Up 1 1.25 Ser/Thr/ CMGC Tyr NEK7 NIMA (never inNM_133494 55, 58, 61 Up 1 1 Ser/Thr/ Other mitosis gene a)- Tyr relatedkinase 7 PACE-1 SCY1-like 3 (S. cerevisiae) NM_020423 56, 59, 62 Up 11.5 Ser/Thr Other CNK cytokine-inducible NM_004073 57, 60, 63 Up 1.25 2Ser/Thr Other kinase (polo-like kinase 3- Drosophila) TTBK tau tubulinkinase 2 NM_173500 84, 87, 90 Up 1.25 1.25 Ser/Thr/ CK1 Tyr PRKCZprotein kinase C, NM_002744 3, 6, 9 Up 2 Ser/Thr AGC zeta TTK TTKprotein kinase NM_003318 64, 67, 70 Up 2.25 Ser/Thr/ Other Tyr ZAP70zeta-chain (TCR) NM_001079 66, 69, 72 Down 1.25 2 Tyr TK associatedprotein kinase 70 kDa FLT3 fms-related tyrosine NM_004119 73, 76, 79Down 1 1 Tyr TK kinase 3 HCK hemopoietic cell NM_002110 74, 77, 80 Down1.5 1.75 Tyr TK kinase BMX BMX non-receptor NM_001721 74, 77, 80 Down1.25 1.5 Tyr TK tyrosine kinase BTK Bruton NM_000061 74, 77, 80 Down 1 1Tyr TK agammaglobulinemia tyrosine kinase DDR2 discoidin domainNM_006182 75, 78, 81 Down 1 1.25 Tyr TK receptor family, member 2 CSF1Rcolony stimulating NM_005211 75, 78, 81 Down 2 Tyr TK factor 1 receptor,formerly McDonough feline sarcoma viral (v-fms) oncogene homolog LCKlymphocyte-specific NM_005356 39, 45, 51 Both 1 1.25 1.5 Tyr GO proteintyrosine kinase PRKCA protein kinase C, NM_002737 1, 4, 7 Up 1 1.25 TyrAGC alpha ROR2 receptor tyrosine NM_004650 10, 13, 16 Up 1 1 1 Tyr TKkinase-like orphan receptor 2 PDGFRA platelet-derived NM_006206 11, 14,17 Up 1 1.25 Tyr TK growth factor receptor, alpha polypeptide SRMSsrc-related kinase NM_080823 11, 14, 17 Up 1.25 1.25 Tyr TK lackingC-terminal regulatory tyrosine and N-terminal myristylation sites TXKTXK tyrosine kinase NM_003328 12, 15, 18 Up 1 1.5 Tyr TK YES1 v-ros UR2sarcoma NM_005433 66, 69, 72 Up 1.25 1.25 Tyr TK virus oncogene homolog1 (avian) DKFZp61P1010 serine/threonin/tyrosine NM_018243 73, 76, 79 Up1.25 1.25 Tyr TK kinase 1 (STYK1) EPHA2 EphA3 NM_004431 73, 76, 79 Up 11.5 Tyr TK FGR Gardner-Rasheed NM_005248 75, 78, 81 Up 1 1.25 Tyr TKfeline sarcoma viral (v-fgr) oncogene homolog FLT1 fms-related tyrosineNM_002019 75, 78, 81 Up 1.25 1.5 Tyr TK kinase 1 (vascular endothelialgrowth factor/vascular permeability factor receptor) EPHB3 EphB3NM_004443 75, 78, 81 Up 1 1.25 Tyr TK CSS3R colony stimulating NM_00521138, 44, 50 Up 2.75 Tyr GO factor 1 receptor, formerly McDonough felinesarcoma viral (v-fms) oncogene homolog GUCY2C guanylate cyclase 2CNM_004963 57, 60, 63 Up 2.25 Tyr Other (heat stable enterotoxinreceptor) BCKDK branched chain NM_005881 10, 13, 16 Down 1.25 1.5 2.25 ?Atypical ketoacid dehydrogenase kinase BRD4 bromodomain NM_014299 11,14, 17 Down 1.75 2 ? Atypical containing 4 AK3 adenylate kinase 3NM_013410 37, 43, 49 Down 1 1.25 X GO FLJ12476 hypothetical proteinNM_022784 39, 45, 51 Down 1 1.25 ? GO FLJ12476 PAPSS2 3-phosphoadenosineNM_004670 42, 48, 54 Down 1 1.5 ? GO 5-phosphosulfate synthase 2C20orf97 chromosome 20 NM_021158 19, 22, 25 Down 2 X CAMK open readingframe 97 (Tribbles homolog 3) C8FW Tribbles homolog 1 NM_025195 19, 22,25 Down 2 X CAMK GS3955 Tribbles homolog 2 NM_021643 20, 23, 26 Down 2 XCAMK FLJ32704 chromosome 9 open NM_157572 37, 43, 49 Down 3.75 X GOreading frame 98 DCK deoxycytidine kinase NM_000788 38, 44, 50 Down 2.25X GO KIAA0626 microfibrillar- NM_021647 39, 45, 51 Down 2.5 X GOassociated protein 3- like XYLB Xylulokinase NM_005108 40, 46, 52 Down 3X GO homolog (H. influenzae) UCK1 uridine-cytidine NM_031432 42, 48, 54Down 2 X GO kinase 1 GUK1 guanylate kinase 1 NM_000858 39, 45, 51 Both 12 2.75 ? GO MGC26954 chromosome 6 open NM_145025 40, 46, 52 Both 1 11.25 ? GO reading frame 199 HK1 hexokinase 1 NM_033498 37, 43, 49 Up2.25 2.75 X GO CALM3 calmodulin 3 NM_005184 38, 44, 50 Up 2 2.25 X GO(phosphorylase kinase, delta) RBSK ribokinase NM_022128 40, 46, 52 Up1.25 1.5 X GO PANK1 pantothenate kinase 1 NM_148978 41, 47, 53 Up 1.51.5 X GO P15RS hypothetical protein NM_018170 41, 47, 53 Up 1 1.5 ? GOFLJ10656 PFKFB2 6-phosphofructo-2- NM_006212 42, 48, 54 Up 1 1.25 ? GOkinase/fructose-2,6- biphosphatase 2 CKMT2 Creatine kinase, NM_00182538, 44, 50 Up 3.5 X GO mitochondrial 2 (sarcomeric) PGK1phosphoglycerate NM_000291 40, 46, 52 Up 2.5 X GO kinase 1

Example 2 Verification of Alpha-Synuclein Phosphorylation Modulation byRe-Screening and by qRT-PCR

The kinases that showed either an increase or decrease inalpha-synuclein phosphorylation from Example 1 were retested to verifythe effect on alpha-synuclein. The confirmation screen was performedusing 10 nM siRNA on the targets identified in Example 1 along withseveral additional kinases of interest. The higher concentration ofsiRNA in Example 1 was used to ensure that marginal knockdown caused bypoorly designed siRNAs could be observed. By using a much lower siRNAconcentration in the confirmation screens, the chance of effects due toa general response to the siRNA itself could be much reduced. SomesiRNAs that were later reported by Ambion to be ineffective were alsore-screened (see replacement library screen below). Finally, some newlyidentified kinases were screened and those results were added to thepool of results. The kinases that were identified as candidates weretested by quantitative RT-PCR (qRT-PCR) to confirm that they wereactually present in the PEAK-Syn cells (see Example 6). The experimentalprocedures and results for the confirmation and rescreening were asfollows:

Confirmation Screen

The results for the confirmation screen were grouped into fourcategories shown below:

Completely Confirmed: This category included the kinases for which allthree siRNAs produced identical phenotypes in the 10 nM screen and inthe 100 nM screen.

Mostly Confirmed: This category included the kinases for which ⅔ of thesiRNAs produced identical phenotypes in the 10nM and in the 100 nMscreen, but one third did not; or, alternatively one siRNA result wasreplicated, but for a second siRNA there was a trend for the samephenotype but with a different siRNA from that used in the originalscreen.

Partly Confirmed: This category included the kinases for which ⅓ of thesiRNAs produced the same phenotype in the 10 nM screen and in the 100 nMscreen.

Not Confirmed: This category included the kinases for which either orboth of the following occurred:

-   -   a) None of the three siRNAs had any effect on        phosphor-alpha-synuclein levels at 10 nM, and/or    -   b) The siRNAs produced the opposite phenotype to what was        observed in the primary 100 nM screen

The number of kinases that fell into each category was tabulated and theresults are shown in Table 3. Seven kinases were completely confirmed,and they are listed in Table 4. Of these seven, only three wereidentified as possessing the qualities to be good candidates for akinase that directly phosphorylates alpha-synuclein at ser-129. This isbecause only three were both ser/thr kinases and decreased phosphoalpha-synuclein levels when the kinase levels were reduced by thespecific siRNA. These included: APEG1, which is believed to play a rolein growth and differentiation of smooth muscle, PLK2 (SNK), which isexpressed in brain and is believed to play a role in normal celldivision, and CDC7L1, a cell division cycle protein with kinaseactivity. Of the three, PLK2 was of the most interest due to its roleand localization in cells, such as activated neurons. Alpha-synuclein isa synaptic-associated protein thought to be involved in synapticplasticity and vesicular transport. Thus, PLK2 was identified as a verygood candidate for a kinase that directly phosphorylates α-synuclein.

TABLE 3 Breakdown of Candidate Hits From 10 nM Confirmation ScreenNumber Hit Category of Hits Completely Confirmed 7 Mostly Confirmed 29Partly Confirmed 22 No Reactivity at 10 nM 19 Opposite Reaction toPrimary Screen 23 Total Number Of Hits Re-Screened at 10 nM 100 NOTES:For all subsequent tables *** denotes where a replacement siRNA has beenanalyzed and the new data substituted for that from the ineffectivesiRNAKey to shading:

TABLE 4 Completely Confirmed Hits

Table 4 shows the seven candidates whose results were completelyreplicated at 10 nM. Only the first three were identified as havingstrong potential to be a direct kinase, because they are ser/thr kinasesthat reduce phospho-synuclein levels when the kinase level is reduced.

There were 29 kinases that fell into the mostly confirmed category, 12of which were candidates for a direct kinase. These are listed in Table5. There were 17 additional kinases that were mostly confirmed at 10 μM.Although not likely to be a direct kinase, these could play a role inthe regulation of a direct kinase and are listed in Table 6. Twenty-twokinases fell into the partly confirmed category. The ser/thr kinasesthat decreased phospho alpha-synuclein (i.e. potentially a direct kinasefor alpha-synuclein) are listed in Table 8, and the remainingpotentially regulatory kinases are listed in Table 8.

TABLE 5 Potential Direct Serine/Threonine Kinases that Mostly Confirmedat 10 nM siRNA

The ser/thr kinases shown in Table 5 were identified as having potentialto be a direct kinase that phosphorylates alpha-synuclein because theysignificantly reduced phospho-synuclein levels when the kinase level wasreduced. 2/3 of the siRNAs produced identical results at 10 nM as theydid at 100 nM, and as such, were designated as Mostly Confirmed hits.

The kinases in Table 6 were designated as Mostly Confirmed, because ⅔ ofthe siRNAs produced identical results at 10 nM and 100 nM concentrationof siRNA. However, because they did not produce the appropriatephenotype or were the wrong class of kinase (i.e. tyr or non-proteinkinase as opposed to a ser/thr kinase), they were identified as notlikely to be a direct kinase that phosphorylates ser-129 onalpha-synuclein. Instead, they may be upstream modulators ofalpha-synuclein phosphorylation.

TABLE 6 Other Kinases That Were Mostly Confirmed at 10 nM siRNA

TABLE 7 Potential Direct Serine/Threonine Kinases that Partly Confirmedat 10 nM siRNA

The ser/thr kinases in Table 7 were identified as having potential to bea direct kinase that phosphorylates alpha-synuclein because theysignificantly reduced phospho-synuclein levels when the kinase levelswere reduced. Only 1/3 of the siRNAs produced identical results at 10 nMas they did at 100 nM, and as such, were designated as Partly Confirmedhits.

Several candidates had contradictory results and, thus, were identifiedas having less potential to be a direct kinase for alpha-synuclein.

TABLE 8 Other Kinases That Were Partly Confirmed at 10 nM siRNA

The kinases in Table 8 were identified as having less potential to bedirect kinases in the phosphorylation of alpha-synuclein at ser-129 butcould be upstream modulators of alpha-synuclein phosphorylation. ⅓ ofthe siRNAs produced identical results at 10 nM as they did at 100 nM,and as such, were designated as Partly Confirmed hits. However, severalhad contradictory results and, thus, were designated as having lesspotential to be direct kinases of alpha-synuclein.

Forty-two kinases did not have their initial results confirmed at 10 nM.Of these, 19 fell into category (a) listed above, and are listed inTable 9. At 10 nM, none of the three siRNAs at 10 nM produced any changein the phospho-alpha-synuclein phenotype, indicating that the resultsfor these kinases from the 100 nM screen were possibly due to off-targeteffects. Twenty-three kinases (Table 10) produced the opposite effect onphospho-alpha-synuclein levels at 10 nM than at 100 nM siRNA. There is apossibility that the results at 10 nM were the true effects due to thefact that at 100 nM results are sometimes masked by off-target effects.This can happen at the much higher siRNA concentration. Alternatively,the true effect may have been seen at the higher concentration. In anycase, these kinases were designated as less likely to be direct kinasesof alpha-synuclein.

The nineteen kinases shown in Table 9 had no significant reactivity at10 nM compared to controls. Thus, it is possible that the change inphospho-synuclein levels observed at 100 nM was due to off-targeteffects caused by high concentrations of siRNA. GPRK5 and GPRK7 were notcandidates in the original 100 nM screen, but were analyzed at 10 nMsiRNA because of additional interest in their role in alpha-synucleinphosphorylation.

TABLE 9 Kinases That Had No Reactivity at 10 nM siRNA

The results for the kinases in Table 10 were not confirmed at 10 nMbecause they had the opposite effect on phospho-synuclein levels fromthat seen at 100 nM. However, it is possible that the results at 10 nMsiRNA were the true results, and that the high concentration (100 nM) ofsiRNA was masking the true effects. It is also possible that the initialeffects observed at 100 nM were the true effects. These were designatedas likely to be direct kinases of alpha-synuclein and set aside to betested further at a later date.

TABLE 10 Kinases Whose Results Were Opposite To The Primary Screen

Replacement and Up-Dated Library Screens

Because some siRNAs used in the initial screen were later identified asbeing of poor quality, screens were performed at both concentrationswith replacement siRNAs. The data for the replacement siRNAs was used toreplace the data for that specific siRNA result from the originalscreen. Statistical data was tabulated for the three siRNAs for eachkinase, and using this, nine additional kinases were identified ascandidates from the original screen that were missed in the primaryscreen. These were retested, and two of the kinases were partiallyconfirmed at 10 nM siRNA. These were BCKDK and FLJ25965 (KSR2).

During the process, a number of new kinases were identified and siRNAsbecame available. These were tested as in Example 1 as an AMBIONUp-Dates library and new kinase candidates were identified. Many of thenewly identified kinases fell under the GO (Gene Ontology Consortium)classification. As such, it was difficult to find detailed informationon some of these kinases. Several of the genes included in this categorywere not true kinases, but were kinase binding proteins or adaptorproteins. At 10 nM siRNA, thirteen kinases were confirmed to becandidates for directly acting on alpha-synuclein. Two of these werelikely candidates for being a direct kinase, see Table 11. The remainingeleven were designated as possible indirect regulators ofphospho-synuclein levels, see Table 11. Table 11 provides Genbankaccession numbers for the kinase sequences as deposited in Genbank as ofNov. 1, 2005.

TABLE 11 Potential Kinase Hits From the Ambion Updates Library

A summary of the results showing the kinase siRNAs that were identifiedand verified in Examples 1 and 2 are shown in Tables 12 and 13. Fromthese results, PLK2, APEG1, CDC7L1, MET, IKBKB, CKII, GRK1, 2, 6 and 7were identified as kinases that are very likely to phosphorylatealpha-synuclein directly or indirectly. The kinases that were identifiedas having siRNAs that increased alpha-synuclein phosphorylation (PRKG1,MAPK13, and GAK) could very well be negative regulators ofalpha-synuclein phosphorylation.

Tables 12 and 13: Summary of Confirmation Studies

TABLE 12

TABLE 13 GRK Results - Mixture of Mostly, Partially and Not Confirmed

In the following examples, in vitro kinase assays were performed on anumber of the potential targets identified in Examples 1 and 2.

Example 3 Identification of Direct Phosphorylation of Alpha-Synuclein InVitro

To determine which of the kinase(s) from the siRNA screen directlyphosphorylated alpha-synuclein, purified kinases were incubated withalpha-synuclein in in vitro kinase reactions. These results showed thatPLK2, GRK2, 5, 6. and 7 (GPRK2, 5, 6 and 7) were all capable ofphosphorylating alpha-synuclein specifically at serine 129 and did notphosphorylate serine 87 in vitro, showing that they could directlyphosphorylate alpha-synuclein. MET, CDC7L1, and IKBKB were shown to beincapable of directly phosphorylating alpha-synuclein (FIGS. 1A-C).

Assay conditions for testing recombinant kinase activities towardrecombinant alpha-synuclein at serine 129 were established and found tobe reproducible by immunoblot and ELISA analyses. Commercially availablerecombinant kinases were used when possible. Those that were notavailable were produced as indicated by recombinant means.

In FIGS. 1A-C, recombinant kinases were included in the in vitroalpha-synuclein (AS) assay by standardizing kinase to alpha-synucleinsubstrate at a constant molar ratio (derived from MW of predicted matureprotein) in each reaction (1:200; kinase: recombinant alpha-synucleinkinase—rAS).—control, +kinase; In FIG. 1A, a probe for totalalpha-synuclein (AS) (mAb Syn-1; 0.1 μg/mL) was used indicatingequivalent substrate in each reaction; In FIG. 1B, a parallel blot wasprobed for S129 phosphorylation (mAb 11A5 1 μg/mL). Prominent signalscame from GRK6, CKI, CKII and PLK2 (not previously tested by activitynormalization). In FIG. 1C, a parallel blot probed for S87phosphorylation (pAb, ELADW-110 5 μg/mL). A signal was detected onlywith CKI phosphorylation.

In FIGS. 1D-F, a more focused study was performed with recombinantkinases from the GPCR-receptor kinase (GRK) family and PLK2 wereincluded in the in vitro alpha-synuclein (AS) assay by standardizingkinase to AS substrate at a constant molar ratio (derived from MW ofpredicted mature protein) in each reaction (1:200; kinase:rAS).—control, +kinase; CAM kinases served as negative controls whileCKI and II served as positive controls. In FIG. 1D, a probe for total AS(mAb Syn-1; 0.1 g/mL) was used indicating equivalent substrate in eachreaction; In FIG. 1E, a parallel blot probed for S129 phosphorylation(mAb 11A5 1 μg/mL). Prominent signals came from all GRKs except forGRK7. A specificity between GRK members could be seen with signal andcan be represented as: CKI>GRK6>PLK2>GRK4>GRK5>GRK2. In FIG. 1F, aparallel blot probed for S87 phosphorylation (pAb, ELADW-110 5 μg/mL).Signal was detected only with CKI phosphorylation.

The assay conditions are defined in Table 14 and were held constant forall kinases tested. All of the kinases listed were available astagged/recombinant protein with the exception of CDC7L1, PRKG1 and APEG.Those putative targets were expressed in an in vitro translation systemand tested in the in vitro AS assay without protein concentration oractivity measurements.

TABLE 14 Assay conditions for in vitro kinase reactions: 1:200 kinase:AS(molarity) total ul Confir- ng kinase; ul in kin. in ng/ul Units/ Totalul ng co- # Kin mation MW kinase 100ul rxn stock kin ul Dilutionkin./rxn kin/rxn factors 1 CKI a′ most 49000 delta 5.1 ng; 6.3 ul 20 8141000 100000x 3 0.024 (1000x (0.01 U/ul) dilution) 2 CKII a′ part 44,000alpha 4.6 ng; 4.3 ul 20 1070 500 50000x 3 0.0642 26,000 beta (1000x(0.01 U/ul) dilution) 3 PAK6 part 38,000 4 ng; 1 ul 50 100 0.21 10x(0.021 1.43 14.3 (100x dilution) U/ul) 4 ARK5 most 78,000 8.1 ng; 8.1 ul50 100 0.06 0.5 50 (100x dilution) 5 CaMK1 most 68,000 7.1 ng; 7.1 ul 50100 0.29 10x (0.029 1 10 calmodulin (100x dilution) U/ul) 1uM 6 PHKG2del 52,000 5.4 ng; 7.7 ul 143 70 0.007 4.3 301 (100x dilution) 7 MAP2K1del 49,000 5.1 ng; 5.1 ul 20 500 1.69 100x 17.75 90 (100x dilution)(0.00169 U/ul) 8 GRK6 part 94,000 9.8 ng; 3 ul 34 290 0.008 3.75 1088(100x dilution) 9 CAMKII del 59,000 6.2 ng; 1.9 ul 31 320 4.93 100x 0.61195 calmodulin delta (100x dilution) (0.0493 1uM U/ul) 10 Met conf50,000 5.2 ng; 5.2 ul 50 100 0.022 1.363 136 (100x dilution) 11 MAPK13conf 46,000 4.8 ng; 1.1ul 22 450 0.054 0.56 250 (100x dilution) 12 PRKG2most 117,000 12.2 ng; 2.8 ul 22 440 0.017 1.76 776 (100x dilution) 13PLK2 conf 106,000 11 ng; 4.1 ul 27 270 0.027 1.1 297 (100x dilution) 14GRK2 most 82,300 8.6 ng; 1.7 ul 20 500 0.0045 6.7 3350 (100x dilution)15 GRK4 part 94,000 9.8 ng; 2.5 ul 25 400 0.0012 25 10,000 (100xdilution) 16 GRK5 part 95,200 9.9 ng; 2.1 ul 21 480 0.00018 167 80,160(100x dilution) 17 GRK7 part 89,700 9.3 ul; 1.9 ul 21 480 0.00067 4521,600 (100x dilution) 18 CDC7L1 conf 63,800 undetermined; nd nd nd ndnd in vitro translation 19 PRKG1 conf 76,200 undetermined; nd nd nd ndnd in vitro translation 20 PDK1 part 59,000 6.1 ng; 3 ul 50 200 0.07410x 4.05 81 SGK (200x dilution) (0.0074 U/ul) 24 APEG conf 12,600undetermined; nd nd nd nd nd in vitro translation

The Standard Conditions were: 40 mM MOPS-NaOH; 1 mM EDTA MgCl 10 mM pH8.0, 0.1% BME; 0.01% Brij-35; 5 ug BSA, 100 uM ATP (5×[substrate]), 100uL volume; 300 ng r-wt-AS (208 nM), (1:200 kinase: AS or activitynormalized 0.03 U/r×n, 34C; 17 hrs. Further, those kinases with varyinglevels of significance/confirmation from combined screening data werepurchased as recombinant, tagged protein, annotated and incorporatedinto a table format for the purposes of establishing in vitro assaysthat were comparable based upon normalization to activity units(determined by the manufacturer from synthetic substrates) orsubstrate:enzyme molar ratios determined from MW and reaction volume.The details of reaction conditions are stipulated in Table 14.Kin.=kinase. For Confirmation: Most=mostly, Part=partially, del=deleted,conf=confirmed.

Kinase activity was initially tested against AS by activity units asdetermined from non-native substrates (peptides or casein). This methodwas used to get a rough estimate as to specificity between kinases andwhether AS was an in vitro substrate for the kinase panel. The resultsof this study are found in FIGS. 2 and 3. At the time of thisexperiment, only a portion of available kinases were obtained and ⅔kinases from the “most probable 7 confirmed” were included (PLK2 was nottested). The most prominent result came from GRK6 (G-protein coupledreceptor kinase 6). CKI gave a modest signal and CKII was notdetectable. Because both CK kinases are known to phosphorylate S129 AS,normalization by activity units was biased against those kinases whichhad higher specific activity for tested substrates vs AS. This waslikely the situation for GRK6 which might have preferred AS as asubstrate rather than the peptide substrate which defined its activityunits.

In the following examples, in vitro kinase assays were performed on anumber of the potential targets identified in Examples 1 and 2. Tocorrect for the activity bias, kinases were retested and newly procuredkinases were put into an assay that normalized for molarity. This gave abetter measurement of stoichiometric ratios between enzyme andsubstrate, thus reporting the phosphorylation event as a function ofAS/kinase interaction. This was in contrast to the event in whichunrelated substrate/kinase phosphorylation was measured. FIGS. 3A-Cillustrate a more realistic view of AS phosphorylation with roughlyequivalent levels of phospho ser-129 between CKI, GRK6 and PLK2 (one of7 highly confirmed). With the exception of CKI, none of the testedkinases were capable of phosphorylating AS at the ser-87 residue. Thisobservation confirmed the specificity/preference of these kinases forthe ser-129 site and/or the low preference/inaccessibility for theser-87 site. However, CKI has been reported to phosphorylate at bothsites.

Effect of Acidic Phospholipid on the Assay Results

The significant levels of activity by GRK6 and PLK2 (polo-like kinasephylogenetically related to the GRK family) in the in vitro assaycombined with the identification of PLK2, GRK2 and GRK1 as decreasers ofphosphorylation in the RNAi screen, prompted a more comprehensive surveyof other GRK members. FIGS. 4A and B indicate the results of GRK 2, 4,5, 6, 7 and PLK2 compared in the in vitro assay. This preference couldbe represented as CKI>GRK6>PLK2>GRK4>GRK5>GRK2. GRK 7 was not able tophosphorylate at appreciable levels. All GRKs were unable tophosphorylate at ser-87 pointing to a specificity for the acidicsequence flanking amino acid 129. These reactions were quantitated andconfirmed by ELISA measurements. These values more or less agreed withthe immunoblot data with an apparent decrease in PLK2 level vs GRKs. Itis likely that most of the AS substrate was depleted (phosphorylated)based on the assay design (300 ng AS, 210 nM for 17 hr).

The positive effect of acidic phospholipids on the phosphorylation of AShas been previously reported Pronin et al. JBC 275(34): 26515-26522(2000) and a pronounced effect on GRK 2 and 5 was observed. Because ofthis report and the many studies indicating that acidic phospholipidsmodulate AS conformation, a mixture of phosphatidylcholine (PC):phosphatidylserine (PS): phosphatidyl-inositol-phosphate-3 (PIP3) wasgenerated and incorporated into the established in vitro assay. Thelipid mixture was shown to increase signal for almost all of the kinasestested. The addition of a lipid environment is likely to imitate themembrane surface in a cell where AS and GRKs are likely to associate.Without being bound by the following theory, it is probably due to afavorable exposure of the C-term of AS upon lipid binding of the N-termhelices of AS. Interestingly, the lipid effect of ser-87 phosphorylation(as see by the CKI reaction) led to a decrease in the level ofphosphorylation. This may be the result of epitope masking by lipidinteraction if ser-87 is buried upon helix interaction.

Example 4 Identification of Direct Phosphorylation of Alpha-Synuclein inCell Lines

Because kinases can be more promiscuous in vitro than they are in cells,an assay was performed in cell lines to confirm the direct interactionwith alpha-synuclein. cDNAs for the kinases that phosphorylatedalpha-synuclein in vitro from Example 3 were transfected into thePEAK-Syn cell line to see which was capable of phosphorylatingalpha-synuclein ser-129 in cells. The results showed that GRK6 and, toan even greater extent, PLK2 were able to mediate alpha-synucleinphosphorylation in cells (FIG. 6).

cDNA clones for PLK2, GPRK6, APEG1, CDC7 and PRKG1 were obtained fromOrigene. The cDNA was transcribed and transfected into PEAK-Syn cellsusing Lipofectamine 2000™ (Invitrogen). For each cDNA analyzed, 12 wellsof a 96-well plate were transfected, along with 12 control wells ofuntransfected cells. Cells were harvested at 48 hrs post-transfection asper the ELISA screening protocol, and analyzed by ELISA for total andphospho-synuclein, and values were normalized for total protein. Forthose kinase targets not commercially available as recombinant proteins(namely APEG, PRKG1 and CDC7LI), an in vitro cell-free reticulocytesystem (Promega) was employed to express protein from human full-lengthcDNA clones (Origene). Proper sequence was determined and DNA prepared.PLK2 and GRK6 cDNA was also included in the study as positive controls.

The percentage of phospho-synuclein in untransfected cells wascalculated to be 7.8%. The percentage of phospho-synuclein for the cellstransfected with APEG1, CDC7 and PRKG1 cDNA was only marginally higherthan untransfected cells at 8.9%. These kinases were considered to haveproduced a negative result in altering phospho-synuclein levels, andwere considered negative controls for experimental purposes, as theywere subjected to the same rigors of transfection that the other kinaseswere exposed to cDNA to PLK2 was transfected into 293-synuclein cells.Cells were harvested 48 hrs following transfection and analyzed by ELISAfor total and phospho-synuclein levels. ELISA values were corrected fortotal protein levels. Overexpression of PLK2 resulted in a dramaticincrease in phospho-synuclein levels, increasing phospho-synucleinexpression by 4.3-fold above expression in untransfected cells.

It is likely that when a direct kinase that phosphorylates α-synucleinis introduced into the cell an increase in phospho-synuclein levelswould be observed. This was the case for both GPRK6 and PLK2 (FIG. 6).The percent phospho-synuclein in cells transfected with GPRK6 cDNAincreased dramatically, from 8.9% to 18.9%. This increase is significantto 9.25 standard deviations above the percent phospho-synuclein observedfor the negative kinases. The increase in phospho-synuclein levels forthe PLK2-transfected cells was even more dramatic, increasing thepercent phospho-synuclein almost four-fold to 33.2%. This represents anextremely significant change, an increase of 22.75 standard deviationsabove the phospho-synuclein levels observed for the negative kinases.This dramatic increase was by far the largest change observed previouslyin using this assay. This data strongly indicates GPRK6, and especiallyPLK2 as very solid contenders as direct kinases responsible forphosphorylating α-synuclein. Thus, as shown in FIG. 6, when GPRK6 cDNAis transfected into HEK-synuclein cells, the expression ofphospho-synuclein increases 2-fold. Introduction of PLK2 cDNA into cellsresults in an even more dramatic increase in phospho-synucleinexpression, a change of almost four-fold above control values.

Example 5 Phosphorylation by PLK2 (SNK) GRK6, CKII and IKBKB

The data in Example 4 was further substantiated for PLK2 by showing thatPLK2 siRNAs reduced alpha-synuclein phosphorylation. This strengthenedthe data showing that PLK2 is a likely candidate as a cellular kinasethat directly phosphorylates alpha-synuclein at Serine 129 (Tables 2 and12).

HEK 293 cells stably transfected with alpha-synuclein were transfectedwith 10 nM and 100 nM of SmartPool siRNAs (Dharmacon). SmartPool siRNAsinclude 4 individual siRNAs to a specific target. Thus, the actualconcentration of each of the four siRNAs transfected into cells was 2.5nM and 25 nM respectively. The results in FIG. 7 show that PLK2significantly decreased phospho-synuclein levels, a change ofapproximately 25%. At 10 nM, but not at 100 nM of siRNA, GPRK6significantly increased the percentage of phospho-synuclein by onestandard deviation above the mean of the control negative kinases (FIG.7). This is the opposite effect to what was previously observed in theprimary siRNA screen and may be due to the quality of the siRNA used inthe first or second assays. These results were confirmed byimmunohistochemistry.

The significant knockdown of phospho-synuclein levels by differentsiRNAs from a different source, independently confirms and solidifiesthe data, and substantiates the role of PLK2 as a direct kinase thatphosphorylates α-synuclein. These experiments were then performed on twoother kinases identified in the screens to be of interest, casein kinasetwo (CKII) and IKBKB.

The individual CKII catalytic subunits were hits in the primary siRNAscreen (see Example 1 and Table 1B) and confirmed at the 10 mM siRNAscreen. It was of interest to determine if the individual CKII subunitsα¹ and α′, when cotransfected with PLK2 or each other, had additiveeffects on alpha-synuclein phosphorylation. Transfections were performedusing the individual CKII subunits A (α¹) and B (α′), cotransfected withPLK2 or each other. Overexpression of these catalytic subunits increasedphospho-synuclein levels by 1.75 and 1 standard deviations respectively(the effect was not additive). When each of the individual subunits wasco-transfected with PLK2, the levels of phospho-synuclein increased overthat of PLK2 alone (18.6% phospho-synuclein) by 1.25 standard deviationseach to 22.8% phospho-synuclein. However when both subunits wereco-transfected with PLK2 phospho-synuclein levels were not significantlyincreased above that for PLK2 alone (21.4% phospho-synuclein).

IKBKB siRNA knockdown resulted in a significant decrease inalpha-synuclein phosphorylation so this gene was tested for capacity tophosphorylate alpha synuclein. Transfections and ELISA analysis wereperformed as per standard procedure. Previous in vitro experimentsdemonstrated that IKBKB was not a direct synuclein kinase as it did notphosphorylate synuclein in a direct kinase assay (see Example 3), butmay be an upstream regulator of synuclein phosphorylation. Thus, IKBKBwas over-expressed in HEK-syn cells to identify the effect onphosphorylation of synuclein. Following introduction of IKBKB cDNA intocells, synuclein phosphorylation increased from 8.3% in the negative(empty vector) control to 21.5%, a 2.6-fold increase. This representedan increase in synuclein phosphorylation that was significant to almost53 standard deviations. The PLK2 positive control increased synucleinphosphorylation to 65.8%, an almost 8-fold increase in phosphorylation(significant to 230 standard deviations). Although the effect onsynuclein phosphorylation was much more modest for IKBKA, a relatedkinase, (1.2 fold) than for IKBKB, it was still significant to 1.4standard deviations.

Example 6 Synphilin as an Alternative Therapeutic Target

Synphilin is a synuclein-associated protein that has been shown to bindalpha-synuclein. To determine if the presence of synphilin can enhancethe phosphorylation of alpha-synuclein, it was over-expressed in HEKcells with and without alpha-synuclein and PLK2. Transfections wereperformed according to standard protocol, followed by alpha-synucleinELISA and analysis. Cells were also harvested for Western blot analysis.Transfected cell lysates were analyzed for total synuclein using 1H7antibody and phospho-serine 129 synuclein using 11A5 antibody (See WO05047860). The total amount of DNA transfected into cells remainedconstant at 0.16 μg/well of a 96-well plate. The type of DNA introducedinto cells varied, with empty vector being used to make up the fullquota of DNA. Varying concentrations of alpha-synuclein, PLK2, andsynphilin cDNA were introduced into naïve HEK cells. Cells transfectedwith all three showed a slight increase in total synuclein. Forphospho-synuclein, the levels in untransfected cells were below thelimit of quantitation. Introducing alpha-synuclein alone yielded 5.2%phospho-synuclein, which was marginally less than co-transfection ofsynuclein with synphilin (5.4% phospho-synuclein). Co-transfection ofPLK2 and synuclein yielded levels similar those observed fortransfecting PLK2 into HEK-syn stable cells, 60% phospho-synuclein.Strikingly, concurrent over-expression of all three cDNA's (PLK2,synuclein and synphilin) resulted in 83.3% phospho-synuclein in the HEKcells. Thus, synphilin increased synuclein phosphorylation in PLK2,alpha-synuclein over-expressed HEK cells.

Increased phosphorylation of alpha synuclein in the presence ofsynphilin can be explained by synphilin binding to the PLK2 polo-boxthereby facilitating phosphorylation of synuclein by PLK2. Synucleinitself is unlikely to bind the polo-box domain.

Example 7 PLK2 Activity: Phosphorylation of Alpha Synuclein and FamilialMutants of Alpha Synuclein

To analyze PLK2 phosphorylation of a number of known familial mutants ofalpha synuclein, in vitro studies were performed and the phosphorylationof the alpha synuclein and mutants analyzed. The familial mutants (FPD)were A30P, A53T, and E46K.

All in vitro reactions were performed using the following conditions, 10mM MgCl2, 100 μM ATP, 27 mM HEPES, 250 ng/ml PLK2, 1/50 dilution ofProtease Inhibitor solution (1 tablet in 1 ml of reaction buffer), 40 mMNitrophenylphosphate, 1 mg/ml of 95% Type II-S Phosphatidylcholine fromsoybean, and 10, 100, or 1000 nM alpha synuclein (AS). The reaction wasincubated at 37° C. The activity was analyzed by autoradiography.

PLK2 was found to be more active against wild-type alpha synuclein thanbeta synuclein. Further, the mutant alpha-synucleins were phosphorylatedmore at a given concentration (especially at lower concentrations) thanWT. A trend of PLK2 activity was identified with PLK2 activity beinghighest with FPD mutants, followed by wild-type alpha synuclein, andminimally against beta synuclein. This order is consistent with amechanism by which phosphorylation of alpha synuclein drives Lewy bodyformation and subsequent pathology.

Example 8 Confirmation of the Presence of Kinases in HEK-Synuclein andSY5Y-Synuclein Cells

qRT-PCR was performed to determine if the kinases of interest wereexpressed in HEK-synuclein and SY5Y-synuclein cells. In Table 15 allsamples were normalized to GAPDH expression. In addition, two of thenegative kinases were analyzed in each experiment as a reference. Of the24 potential direct kinase candidates tested, 20 were detected in theHEK293-synuclein cells, including PLK2. Thus, the remaining completelyconfirmed kinases were detected in the cells (FIG. 9). Four of thepotential direct kinases tested, GPRK1, GPRK7, ERK8 and RIPK3, were notdetected. GPRK6 was barely detectable.

The qRT-PCR was performed as follows: the mRNA levels were normalized toGAPDH mRNA expression levels. Total RNA was purified from a cell pelletusing the QIAGEN RNeasy Kit and protocol. Primer-probe sets for 24 ofthe potential direct kinases and the four indirect completely confirmedkinases were ordered from Applied Biosystems (TaqMan Gene ExpressionAssays), along with reverse transcriptase, RNase inhibitors and standardPCR reagents. A one-step RT-PCR/qRT-PCR reaction as performed an ABI7500Real-Time PCR machine for each primer-probe set using 20 ng or 200 ngtotal RNA using the following cycling conditions: 48° C./30 mins (RT-PCRstep), 95° C./10 mins (denature), then 40 cycles of 95° C./15 secs, 60°C./1 min. For each primer-probe set, an RT-negative reaction andPCR-negative reaction was performed. The RT-negative controls forbackground amplification of DNA (not RNA) that is contaminating thepurified RNA. The PCR-negative control was to ensure all of the PCRreagents were free of contaminating RNA and DNA, and should have had nosignal.

All three of the completely confirmed potential direct kinases, alongwith the four indirect completely confirmed kinases were easily detectedin SY5Y-synuclein cells, indicating this cell line may be a viableoption for a neuronally-derived cell line for further experimentalanalysis of kinases.

TABLE 15 qRT-PCR demonstration of the presence of kinases inHEK-synuclein and SY5Y-synuclein cells Sample Name Relative Expression(to GAPDH) SYN APEG1 2.23 SYN SNK/PLK2 3.57 SYN CDC7L1 1758.34 SYN PRKG17.36 SYN MAPK13 8.97 SYN GAK 2.17 SYN MET 891.44 SY5Y-SYN APEG1 1698.45SY5Y-SYN 208.66 SNK/PLK2 SY5Y-SYN CDC7L1 24.42 SY5Y-SYN PRKG1 42.22SY5Y-SYN MAPK13 45.73 SY5Y-SYN GAK 17.63 SY5Y-SYN MET 86.22

Example 9 Identification of Increased Phosphorylation of Alpha-Synucleinin 293 Cells and Neuronally-Derived Cell Lines

PLK2 and GRK were overepressed in 293 cells stably transfected withalpha synuclein. ELISA and Western blot were performed to identifyincrease in phospho-synuclein with PLK and GRK kinases and an increasein phosphorylation was demonstrated. A second method was used to confirmthe increase using the same biotinylated antibodies used in the ELISAfor immunostaining (11A5) in 293 cells. This method also demonstrated anincrease in phospho-synuclein in cells transfected with PLK2 and to alesser extent GRK. The increase was detected in a small population ofcells that brightly stain for 11A5, not a general increase in all cells.The amount of total synuclein (measured using the 5C12 antibody) did notappear to change. This was a significant increase in phosphorylation inthe 293 cells. Thus, it was of interest to see if the results could berepeated in neuroblastoma cells.

To identify that the dramatic upregulation of phospho-synuclein observedwith PLK2 and GPRK6 occurs in neuronally-derived cells, the sameexperiment was performed in human neuroblastoma cells (SY5Y cells).Immunostaining results showed that PLK2 caused an increase in thephospho-synuclein in a small population of cells, in a very similarpattern to the 293 cell experiments. Quantitation was performed byimmunohistochemistry using the ArrayScan™ in two ways. First all cellswere counted and did not show any difference. Then just the bright cellswere counted and this analysis showed about a 5-10 fold increase in thenumber of 11A5 positive cells that were PLK transfected, with a slightincrease with GRK6 as well.

The cDNA transfection experiment is repeated in HCC cells andimmunohistochemistry is performed with a variety of alpha-synucleinantibodies on the cells that have been transfected with PLK2 and GPRK6.Cells may be treated with additional reagents to mimic the pathology ofParkinson's disease; such reagents could include, for example, rotenone,paraquat, hydrogen peroxide, or ferric chloride. In this way, inclusionformation and/or alpha-synuclein aggregation is observed in these cells.Antibodies used to look for inclusions/aggregation include LB509, SYN-1,11A5 and ELADW-110.

Next, cDNA for PLK2 and GPRK6 siRNA is transfected in primary neuronalcultures in preparation for introducing targets into a mouse model. Themethod is performed as in Example 4. qRT-PCR is performed (as in Example2) using SYSY-synuclein RNA. SY5Y-synuclein cells are derived fromneuroblastoma cells and have been stably transfected with a WT-synucleinvector.

Example 10 Distribution of Lentivirus-Expressed Alpha-Synuclein in HumanCortical Culture (HCC)—a Cellular Model for Lewy Body Disease

Of interest was the identification of a cellular model for Lewy bodydisease and/or for PD pathology. Thus, lentivirus-mediated expression ofalpha-synuclein in human cortical cultures was used to establish a modelof alpha-synuclein deposition in vivo. Experiments were performed ondonors, and HCC cells overexpressing wild-type and variantalpha-synuclein to fractionate the cells and localize wild-typealpha-synuclein and variant alpha-synuclein within the cells. In oneexperiment aggregation of alpha synuclein in a manner matching LBdisease was observed in the HCC cells. Further, in one experiment whenPLK2 was expressed, the phosphorylation of alpha-synuclein as well asthe aggregation increased. In other experiments this was not observed.

Further experiments were performed to determine whether extendingculture might increase the accumulation of overexpressed synuclein, andwould stress the cells, which also might favor synuclein deposition ortoxicity. Accordingly, HCC were transduced with viral vectors expressingWT, A53T, S129A or both A53T/S129A alpha-synuclein mutants. Followingtransfection cells were grown in vitro for 9, 16 or 23 days beforecollecting and fractionating. ELISA results were normalized to proteinconcentration and showed an accumulation of synuclein in the solublefraction with increasing time. Somewhat greater accumulation wasobserved with the S129A mutant.

When further experiments were performed with WT, 119-truncated, and E46KAS, the results were as follows. The higher the expression of wild-typethe larger the portion of alpha synuclein recovered in the solublefraction. E46K synuclein showed a 50-100% increase in the amount ofphosphorylated alpha-synuclein. However, the E46K mutation did notmarkedly affect relative amounts of synuclein recovered in themembrane-bound or insoluble fractions. Expression of 119-truncated alphasynuclein led to a slight increase in the relative amount accumulatingin the insoluble fraction (about 3 fold higher relative to WT). Theincrease is expected in view of the published results suggesting thattruncated synuclein forms fibrils much more readily in vitro than doesfull-length (Murray et al. 2003 Biochemistry 42:8530). The 119truncation resulted in an increased association with membranesconsistent with the N-terminal domain being responsible for associationwith lipid bilayers. The increased association with membranes maymitigate the increased tendency of the soluble protein to aggregate. Theresponse of the insoluble fraction to increases in levels of solublesynuclein on overexpression and to truncation, a change favoringaggregation, suggest that it provides a way to identify factorsaffecting aggregation in the intraneuronal milieu.

The increased alpha-synuclein in the soluble compartment might beshifting the alpha-synuclein to a potentially more vulnerablecompartment, leading to changes which could result in increaseddeposition. Since the kinases proposed to phosphorylate alpha-synucleinat Ser129 are soluble, it seems likely that the soluble alpha-synucleinis more accessible to phosphorylation as well.

Additional experiments are performed to identify inhibitors of thephosphorylation and/or aggregation in this cellular model by expressingthe inhibitors in the cells and identifying a reduction in thephosphorylation and/or aggregation.

Example 11 Analysis of Endogenous Kinase Activity in Alpha-SynucleinKnock-Out Mice

The utilization of an alpha-synuclein knockout (alpha-synuclein KO)mouse brain for the identification of a putative alpha-synuclein kinasehas the advantage over the siRNA screen in the following ways: 1) theuse of brain material provides relevant and possibly higher levels ofbrain-specific kinase activity which the HEK cell line may not provide;2) cofactors may be present in brain (lipid, protein, etc.) which maynot be present in cells and 3) absence of any endogenous alpha-synucleinwhich could be detected as a phosphorylated AS. The inclusion of 25-50μg of extracts (soluble and detergent soluble) with recombinantalpha-synuclein (rAS) was assessed with 250 μM ATP to determine ifappreciable kinase activity was present in crude material. FIG. 8A showstotal alpha-synuclein in each reaction indicating equivalent loadings ofrAS. In FIG. 8B, the levels of phospho-ser-129 alpha-synuclein wereinvestigated. The rAS in both TBS (sucrose soluble) and TX (Triton-S 100soluble) extracts was phosphorylated, with roughly twice the level ofsignal from the TBS material than the TX (although reactions were notnormalized for protein). Phosphorylation levels were increased byaddition of CKI but were not significantly affected by the addition ofphospholipids from soybean. An identical blot was probed for ser-87phosphorylation in FIG. 9B. This pAb presents cross-reactivity with rASat 100 ng, thus levels above background indicate true phosphorylation atthe ser-87 site. In both TBS and TX reactions there is no significantphosphorylation at this site whereas the CKI spike achievedphosphorylation at appreciable levels. These experiments suggest thatmeasurable and real kinase activit(ies) are present in the soluble andmembrane fractions of KO mouse brain and are specific to the ser-129site compared to ser-87. The potential exists for phosphorylation atother serine or threonine sites in alpha-synuclein but antibodies arenot yet available to detect such modifications. Thus, measurable levelsof ser-129 specific kinase activity/activities are present inalpha-synuclein KO mouse brain extracts and could serve as startingmaterial for purification of a kinase from the brain.

In FIGS. 8A, 8B, 9A and 9B, cortices of alpha-synuclein KO mouse brainwere Dounce homogenized to obtain 200 mM sucrose soluble and 0.1% TritonX-100 soluble extracts with protease and phosphatase inhibitors present.20 μl of sample (100 μl total volume of reaction) was incubated with 2.4μg of wt-rAS in the presence or absence of 1000 units of casein kinaseI(CKI) as a positive control, and/or 200 μg of phosphatidycholine (PC;soybean lecithin) to increase kinase activiti(es). Reactions were loadedon SDS-PAGE (130 ng total AS) and immunoblotted with Syn-1 (total Syn;0.1 ug/ml), 11A5 (phospho ser-129; 1 μg/ml) or ELADW110 (phospho ser-87;2 μg/mL).

The above data shows that PLK2 other direct and/or indirect kinases(such as GRK6), and modulators such as synphilin are novel targets fortherapeutic intervention in DLB and PD. PLK2 is a preferred targetbecause it can directly phosphorylate alpha-synuclein specifically atser-129.

Example 12 Effect of Overexpression of PLK Family Members on SynucleinPhosphorylation

Overexpression of PLK family members PLK1, 2 and 3, but not PLK4,increased synuclein phosphorylation above the level of the endogenouskinase in HEK-293 cells.

Methods

HEK-293 naïve cells were transfected with an expression vector encodingPLK1, PLK2, PLK3, or PLK4 under the control of a CMV promoter, or emptyvector, together with an expression vector encoding WT-synuclein. Allvectors had the sequence coding for the protein of interest.Transfection was accomplished using Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.) with 0.08 μg vector. Cells were washed and harvested48 hours post-transfection. A Micro BCA (Pierce) total protein assay andtotal synuclein and phospho-synuclein ELISAs were performed on eachplate of treated cells.

Results

Results are summarized in Table 16.

TABLE 16 Proportion of Fold Change total synuclein that in Percent isphoshorylated at ser-129 Synuclein Endogenous Kinase 2.0%Phosphorylation Endogenous Kinase + 1.6% 0.8 synphilin Over-ExpressedPLK1 4.1% 2.1 Over-Expressed PLK2 42.3% 21.2 Over-Expressed PLK3 79.2%39.6 Over-Expressed PLK4 1.9% 1.0

PLK2

Over-expression of PLK2 in the 293 cells yielded similar results tothose observed in previous experiments, resulting in a 21-fold increasein synuclein phosphorylation, from 2% to 42.3%.

PLK3

The over-expression of PLK3 generated an even more striking 39-foldincrease in synuclein phosphorylation, from 2% to 79.2%phospho-synuclein. However, knockdown of PLK3 does not decrease thepercentage of phosphorylated synuclein in the 293 cells (see Example 13,infra). PLK3 is structurally most similar to PLK2 of all the PLK familymembers, so even if PLK3 is not the synuclein kinase, it may be able toperform the same physiological tasks as PLK2. It has been proposed thatin the absence of PLK2 expression, PLK3 can functionally compensate forthe absent PLK2 (Smith et al., 2006, “Epigenetic inactivation implies atumor suppressor function in hematologic malignancies for Polo-likekinase 2 but not Polo-like kinase 3.Cell Cycle.” Cell Cycle 5:1262-4).In addition, over-expression experiments can yield off-target andnon-physiological effects and results should be substantiated throughadditional experimentation such as siRNA knockdown or in vitrotranscription/translation experiments.

PLK1

Over-expression of PLK1 resulted in a two-fold increase in the percentof synuclein phosphorylation (from 2% to 4.1%). While not as robust anincrease in synuclein phosphorylation as the 21-fold increase with PLK2or 39-fold increase with PLK3 over-expression, it is still significant.However, as noted above, overexpression of proteins can yield resultsthat do not accurately represent the physiological state within cellsand tissues. Over-expression of genes can produce off-target effects andcan result in erroneous localizations within cells that do notcharacterize the true physiological state, and results ofover-expression experiments should be substantiated with additionalexperimentation such as siRNA knockdown or in vitrotranscription/translation experiments.

PLK4

Over-expression of PLK4 did not change the percentage of synucleinphosphorylation in 293 cells. The structure of PLK4 is much differentfrom those of the other PLKs, and has only a single polo box domainrather than the two that the other three family members have. Theresults do not exclude the possibility that in other tissue types, PLK4may be able to phosphorylate synuclein.

In Vitro Biochemical Assays

In vitro biochemical assays using each of the four PLK family members asthe kinase to phosphorylate synuclein were conducted. The results (notshown) mirror the cell-based overexpression assays described above, withPLK1 being able to phosphorylate synuclein moderately, PLK2 and PLK3having extremely robust phosphorylation of synuclein, and PLK4exhibiting no ability to phosphorylate synuclein.

Example 13 siRNA Knockdown of PLK Family Members

HEK-293 naïve cells were transfected with 40 nM, 100 nM and 200 nM ofDharmacon On Target Plus Smart Pool siRNAs (Darmacon, Lafayette, Colo.)designed to knock down the expression of each of the four PLK familymembers. The transfection was performed using Lipofectamine 2000(Invitrogen Carlsbad, Calif.). Cells were washed and harvested 48 hourspost-transfection. A Micro BCA (Pierce) total protein assay and totalsynuclein and phospho-synuclein ELISAs were performed on each plate oftreated cells.

PLK2

The results of the siRNA knockdown experiment are summarized in FIG. 10.In agreement with what we have observed previously for PLK2 siRNAinhibition (see Example 5) knockdown resulted in a 25% decrease in thepercentage of phosphorylated synuclein in the 293 cells.

PLK3

The knockdown of PLK3 also had no effect on synuclein phosphorylation,which is not in agreement with the PLK3 overexpression data. This may bedue to the fact that overexpression can be somewhat promiscuous, and notindicative of the true physiological state within cells. It may also bethat PLK3 is not the synuclein kinase in 293 cells, but it could stillbe the synuclein kinase in neurons or other cells due to the potentialfor isoform switching between cell types. Thus, although PLK3 knockdowndoes not reduce synuclein phosphorylation as we would expect frominhibition of the synuclein kinase, it does not completely eliminatePLK3 as being the correct PLK family member as the synuclein kinase inneurons.

PLK1

Cells treated with PLK1 siRNA showed a 40% decrease in the total proteinlevels compared to the negative siRNA control, indicating that treatmentof cells with PLK1 siRNA inhibits proliferation of cells. This has beennoted in the literature, and confirms the role of PLK1 in mitosis.Taking into account this change in total protein levels, knockdown ofPLK1 transcript results in a 60-90% increase in the percentage ofphospho-synuclein. This indicates that PLK1 is negative regulator ofsynuclein phosphorylation. It appears that when PLK1 is present in cell,it regulates PLK2 or it's upstream pathway and accordingly the level ofPLK2-mediated phosphorylation of synuclein. This increase inphospho-synuclein with PLK1 knockdown has been observed in twoindependent experiments, and is intriguing as a potential regulator ofPLK2-driven synuclein phosphorylation.

PLK4

Knockdown of PLK4 with siRNA had no effect on synuclein phosphorylation,in accord with the PLK4 overexpression data above (Example 11).

Example 14 Treatment of Primary Neuronal Cultures with Kinase Inhibitors

The effect on levels of serine-129 synuclein was tested for kinaseinhibitors with various specificities in rat and mouse primary corticalcell culture was tested.

Table 17 shows the inhibitors used in the experiment:

TABLE 17 Inhibitor Primary Specificity 1 ELN-481080 PLK1 2 ELN-481574-2(BI 2536) PLK 1, 2, 3 3 ELN-481530 JNK3 4 DMAT (2-Dimethylamino-4,5,6,7-Casein kinase 2 tetrabromo-1H-benzimidazole) 5 Scytonemin PLK1, PKCβ1,PKCβ2, Cdk1/B, Myt1, and Chk1 6 K252A Generic kinase inhibitor 7Wortmannin Generic kinase inhibitor 8 N-benzoyl staurosporine Generickinase inhibitor 9 TBB (4,5,6,7-Tetrabromo-2- ATP/GTP-competitiveazabenzimidazole) inhibitor of casein kinase 2

Preparation of Mouse Neuronal Cell Cultures

Mouse cortical cultures from fetal (i) Swiss-Webster, (ii) C56BL/6 WTand (iii) C56BL/6 E46K-Synuclein Transgenic mice were prepared andmaintained in B27/DMEM/1% Penicillin-Streptomycin at 37° C./10% CO₂ forthree to fourteen days and then treated with kinase inhibitors.

Cultures were exposed to inhibitors for two hours in B27/DMEM/1%Penicillin-Streptomycin. Cells were immediately washed in 100 μL of PBSplus Mg²⁺ and Ca²⁺, and harvested in ice-cold CEB minus EGTA plusprotease inhibitors, frozen on dry ice and stored at −80° C. untilprocessed. A Micro BCA (Pierce) total protein assay, and total synucleinand phospho-synuclein ELISAs were performed on each plate of treatedcells using standard methods.

Preparation of Rat Neuronal Cell Cultures

Rat Ventral Mesencephalon (RVM) cultures were prepared from E15 Wistarrats. The RVM cultures were cultured for 2 days and transduced with 0.75MOI E46K-Synuclein lentivirus alone, or 0.75 MOI E46K-Synucleinlentivirus plus 0.75 MOI ca-PLK2 lentivirus. The mid-brain region,ventral mesencephalon, from embryonic day 15 Wistar rats were dissectedand pooled for processing for culture as described previously in Stevenet al., 2001, Genetics 10:1317-24 with the medium supplement replacedwith B27 (Invitrogen) and 1% FBS (HyClone). Transduced cultures weremaintained in neuronal media with B27/1% FBS and 37° C./5% CO₂ untiltreated with inhibitors. On DIV 17 (which corresponds to 15 days afterviral transduction of the cultures) cells were treated with inhibitorsfor two hours. Cells were then harvested as detailed above in neuronalmedia with B27/1% FBS. A Micro BCA (Pierce) total protein assay, andtotal synuclein and phospho-synuclein ELISAs were performed on eachplate of treated cells using standard methods.

Results

The effects of inhibitors on phosphorylation of alpha-synuclein aresummarized in Tables 18 and 19. Table 18 shows that the extent ofinhibition of synuclein phosphorylation by selected inhibitors issimilar for the endogenous kinase in mouse and rat primary cultures, aswell as a constitutively active PLK2 variant (caPLK2). In caPLK2 thepolo-box has been deleted, activating the Thr to Asp mutation in thekinase activation loop. Table 19 shows the rank order of potency forselected inhibitors is similar between cell types and across species(mouse, rat and human)

The results of treatment of several primary neuronal cultures with PLKand other inhibitors were very similar to those observed in 293 cells inboth the EC₅₀ values (Table 18) and the rank order of potency (Table19). The EC₅₀ values for the potent PLK inhibitor BI 2536 (ELN-481574-2)in all cellular paradigms were in the nanomolar range, with most EC₅₀values being 100 nM or less, and has an IC₅₀ of 27 nM in the in vitrobiochemical assay. This PLK inhibitor is a very potent inhibitor ofsynuclein phosphorylation in primary neuronal cultures in mouse and rat,substantiating the role of a PLK family member as the synuclein kinase.

The second most potent inhibitor in most of the cellular paradigmstested was the generic kinase inhibitor K252A (EC₅₀ values of 3-7 μM).In the in vitro biochemical reaction, K252A does not inhibit PLK2-drivensynuclein phosphorylation (IC₅₀>10 μM), indicating that its inhibitionof synuclein phosphorylation is through an upstream or downstreamregulator of PLK2.

The third most potent inhibitor of synuclein phosphorylation in primaryneuronal cells is ELN-481080, a PLK1 inhibitor that also has inhibitoryactivity on PLK2, and to a lesser degree, PLK3 and PLK4 (see Table 21).The IC₅₀ of ELN-481080 was 7.7 μM, and the EC₅₀ values in most cellularparadigms was 12-38 μM.

The fourth most potent inhibitor of synuclein phosphorylation in primaryneuronal cells is the casein kinase 2 inhibitor DMAT(2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole). The EC₅₀ valuesfor inhibition of synuclein phosphorylation in each of the cell typeswere 16-33 μM. In the biochemical assay DMAT was the second most potentinhibitor of synuclein phosphorylation with an IC₅₀ of 2.07 μM. ThatDMAT can directly act on PLK2 to inhibit synuclein phosphorylationsuggests that reports by others using to DMAT to show that the CKII isthe synuclein kinase may be due to the inhibitory effect the DMAT has onPLK2 and not on casein kinase 2. To confirm that the inhibitory effectDMAT is due to inhibition of PLK2, MCC were treated with TBB, a veryspecific CKII inhibitor. TBB had no effect on synuclein phosphorylation,even at concentrations of 100 μM (data not shown). Thus, we areconfident that the inhibitory effect of DMAT on synucleinphosphorylation is due to its direct activity on PLK2 and not CKII.

The other four inhibitors tested, Scytonemin, Wortmannin and N-benzoylstaurosporine did not inhibit 50% of synuclein phosphorylation in any ofthe cells or in the in vitro assay. ELN-481530, the JNK3 inhibitor, alsodid not inhibit PLK2-driven phosphorylation in vitro, or in most celltypes tested. However, in RVM stably transduced with E46K-synuclein (butnot in conjunction with caPLK2), concentrations of 1 μM or greater ofELN-481530 and above inhibited 50% of synuclein phosphorylation, withthe inhibition reaching a plateau of 50%. In addition, 293 cellsover-expressing human WT-synuclein and WT-PLK2 reached the same plateauof 50% inhibition at 10 μM ELN-481530. While further work needs to bedone to elucidate the role that JNK may play in synucleinphosphorylation, it seems reasonable to suggest the JNK may be aregulator of PLK2 and synuclein phosphorylation in certain cell types.

TABLE 18 The EC₅₀/IC₅₀ values for selected inhibitors are similarbetween cell types and across species (mouse, rat and human) RVM E46K-Over- Straight E46K-Syn TG Swiss-Webster RVM E46K- Synuclein EndogenousExpressed Inhibitor Biochemical WT-MCC MCC MCC Synuclein caPLK2 Kinase(293) PLK2 (293)* ELN-481080 7.7 μM 17.3 μM 12.3 μM 14.8 μM ~100 μM 25.2μM ~100 μM 38 μM ELN-481574-2 0.027 μM <0.1 μM <0.1 μM 0.092 μM 0.598 μM0.078 μM 0.054 μM 0.07 μM DMAT 2.07 μM 18.7 μM 16.6 μM 21.6 μM 20 μM28.5 μM 33 μM 18.6 μM K252A >10 μM 3 μM 3.1 μM 2.9 μM 3.2 μM 3.5 μM 3.6μM 7.1 μM ELN-481530 >100 μM >100 μM >100 μM NT ~1 μM >100 μM >100 μM~10 μM Scytonemin >100 μM >100 μM >100 μM NT >100 μM >100 μM >100μM >100 μM Wortmannin >100 μM >100 μM >100 μM NT >100 μM >100 μM >100μM >100 μM N-Benzoyl >100 μM >100 μM >100 μM NT >100 μM >100 μM >100μM >100 μM Staurosporine *See Example 14 NT - Inhibitor not tested inthese cells

TABLE 19 The rank order of potency for selected inhibitors is similarfor different cell types and species (mouse, rat and human) RVM E46K-Over- Straight E46K-Syn TG Swiss-Webster RVM E46K- Synuclein EndogenousExpressed Inhibitor Biochemical WT-MCC MCC MCC Synuclein caPLK2 Kinase(293) PLK2 (293)* ELN-481080 3 3 3 3 5 3 4 5 ELN-481574-2 1 1 1 1 1 1 11 DMAT 2 4 4 4 4 4 3 4 K252A 4 2 2 2 3 2 2 2 ELN-481530 NA NA NA NA 2 NANA 3 Scytonemin NA NA NA NA NA NA NA NA Wortmannin NA NA NA NA NA NA NANA N-Benzoyl NA NA NA NA NA NA NA NA Staurosporine *See Example 14NA—Not Applicable due to an EC₅₀ not being reached

Example 15 Specificity of ELN-481574

PLK inhibitor ELN-481574-2 (BI 2536) exhibited high potency for reducingalpha-synuclein phosphorylation in a variety of cells, including primaryneuronal cells (see Example 14). The inhibitor was screened against apanel of 260 kinases (almost half the kinome) and found that at 10 uM,ELN-481574 was very potent in inhibiting PLK2 and PLK3, although thecompound also inhibited several CaMKs and casein kinases. The tenkinases that were inhibited the most by ELN-481574 were subjected to anine-point dose-response (0.003 uM to 3 uM) of the compound to determinethe IC₅₀. The ELN-481574 compound has 16-fold selectivity for PLK2 (IC₅₀11 nM) and 13-fold selectivity for PLK3 (IC₅₀ 14 nM) over the nextclosest kinase IC₅₀ (CaMKIIδ 182 nM). See Table 20 for a summary. Thisconfirms that this inhibitor is indeed potent and highly selective forat least two members of the PLK family.

TABLE 20 Summary of IC₅₀ Values for ELN-481574-2 Kinase IC50 (nM) PLK211 PLK3 14 CaMKIIδ 182 FAK 239 EGFR(L858R) 264 Fes 341 MLCK 643 PKCμ1,126 CK1γ3 1,231 CaMKIIβ 1,496

Example 16 Effect of Kinase Inhibitors in HEK-293 Cells OverexpressingPLK Family Members

HEK-293 cells as described in Example 12, which overexpress synuclein inconjunction with empty vector or one of the PLK family members, wereexposed to inhibitors. EC₅₀ values (Table 21) and a rank order ofpotency (Table 22) was similar to that seen in primary neuronal cells(see Example 13). The PLK inhibitor ELN-481574-2 was the most potentinhibitor of synuclein phosphorylation by the endogenous kinase and byeach of the over-expressed PLKs. The EC₅₀ for the endogenous kinase was99 nM, and was 36 nM, 80 nM, 438 nM and 192 nM for each of the PLK1, 2,3 and 4. The EC₅₀ for PLK3 is somewhat higher than it is for theendogenous kinase or the other PLKs.

The second most potent inhibitor of synuclein phosphorylation was DMAT,with EC₅₀ values of ˜14-74 μM for the over-expressed PLKs and 56 μM forthe endogenous kinase. The PLK1 inhibitor ELN-481080 shows selectivityfor PLK1 (16.7 μM) over PLK2 (47.8 μM), and while an EC₅₀ was notreached for inhibition of synuclein phosphorylation by over-expressedPLK3 or PLK4, ELN-481080 did inhibit 30-40% of synuclein phosphorylationat concentrations of 30-100 μM. It should be noted that while the EC₅₀for ELN-481080 activity on the endogenous kinase was >1100M in thisexperiment, in previous experiments, the EC₅₀ has been ˜100 μM (Table18), which is still within the three-fold range of variation of the EC₅₀of PLK2.

In this experiment, treatment of overexpressed PLK family members withthe JNK3 inhibitor ELN-481530 did not inhibit synuclein phosphorylationby 50%. However, it did inhibit 30-45% of synuclein phosphorylation bycells overexpressing PLK2 or PLK3 at concentrations of 1 μM and above.In the presence of endogenous kinase or over-expressed PLK1 or PLK4,inhibition was lower, reaching a maximum of 15-20%.

TABLE 21 The EC₅₀ values for ELN-481574-2 is similar for the four PLKfamily members EC₅₀ of Selected Inhibitors Based on the PercentInhibition of Synuclein Phosphorylation Inhibitor Synuclein + VectorSynuclein + PLK1 Synuclein + PLK2 Synuclein + PLK3 Synuclein + PLK4ELN-481080 >100 μM 16.7 μM 47.8 μM >100 μM >100 μM ELN-481574-2 0.099 μM0.036 μM 0.08 μM 0.438 μM 0.192 μM DMAT 55.7 μM 27.2 μM 13.9 μM 37.3 μM74.3 μM ELN-481530 >100 μM >100 μM >100 μM >100 μM >100 μM

TABLE 22 The rank order of potency of inhibitors tested is very similarbetween PLK family members Rank Order of Potency of Inhibitors Based onPercent Inhibition of Synuclein Phosphorylation Inhibitor Synuclein +Vector Synuclein + PLK1 Synuclein + PLK2 Synuclein + PLK3 Synuclein +PLK4 ELN-481080 NA 2 3 3 NA ELN-481574-2 1 1 1 1 1 DMAT 2 3 2 2 2ELN-481530 NA NA NA NA NA NA—Not Applicable due to an EC₅₀ not beingreached

Example 17 Effect of Staurosporin in HEK-293 Cells Overexpressing PLK2

HEK-293 cells as described in Example 12, which overexpress synuclein orsynuclein and PLK2. The EC₅₀ value for the endogenous kinase (synucleinonly) was 4.35 μM, and 11.16 μM for PLK2 over-expressing cells.

The above examples are illustrative only and do not define theinvention; other variants will be readily apparent to those of ordinaryskill in the art. The scope of the invention is encompassed by theclaims of any patent(s) issuing herefrom. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theissued claims along with their full scope of equivalents. Allpublications, references (including accession numbers), and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were so individually denoted.

1. A method for inhibiting phosphorylation of alpha-synuclein in amammalian cell, the method comprising reducing polo-like kinase 2 (PLK2)activity in the cell such that phosphorylation of synuclein is reduced.2. A method for inhibiting phosphorylation of alpha-synuclein in amammalian cell, the method comprising contacting the cell with acompound that reduces PLK2 activity in the cell such thatphosphorylation of alpha-synuclein is reduced.
 3. The method of claim 2wherein the agent reduces expression of the PLK2 protein.
 4. The methodof claim 2 wherein the cell is a neuronal cell.
 5. The method of claim 4wherein the agent has a molecular weight less than
 4000. 6. The methodof claim 5 wherein the agent is a synthetic compound.
 7. The method ofclaim 4 wherein the agent preferentially reduces PLK2 activity activityrelative to reduction of PLK1 activity, PLK2 activity, or PLK3 activity.8. The method of claim 7 wherein the agent is a polynucleotide thatinhibits expression or translation of a PLK2 RNA transcript.
 9. Themethod of claim 8 wherein the agent is an siRNA comprising a doublestranded region.
 10. The method of claim 9 wherein one strand of thedouble stranded region is perfectly complementary to a PLK2 transcriptand lacks perfect complementarity to a PLK1 transcript or a PLK3transcript.
 11. The method of claim 5 wherein the compound is4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pteridinyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide.12. A method of treating a patient diagnosed with Parkinson's Diseasecomprising administering a therapeutically effective amount of an agentthat inhibits PLK2 activity.
 13. The method of claim 12 wherein theagent preferentially inhibits PLK2 activity relative to inhibition ofPLK1 activity or PLK3 activity.
 14. The method of claim 13 wherein theagent preferentially inhibits PLK2 activity relative to inhibition ofPLK1 activity and PLK3 activity.
 15. The method of claim 14 wherein theagent is an siRNA.
 16. The method of claim 15 wherein the agent is ansiRNA comprising a double stranded region.
 17. The method of claim 16wherein one strand of the double stranded region is perfectlycomplementary to a PLK2 transcript and lacks perfect complementarity toa PLK1 transcript or a PLK3 transcript.
 18. The method of claim 12wherein the agent is a synthetic compound with a molecular weight lessthan
 4000. 19. The method of claim 12 wherein the compound is4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pteridinyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide.20. The method of claim 12 wherein the patient is not diagnosed or undertreatment for cancer.
 21. The method of claim 12 wherein the patient isnot diagnosed or under treatment for Alzheimer's disease.
 22. A methodof identifying an agent reduces alpha-synuclein phosphorylation in amammalian cell expressing alpha-synuclein comprising selecting an agentthat a) reduces activity of PLK2 in a cell expressing PLK2; and b) doesnot reduce activity of PLK1 in a cell expressing PLK1, or reducesactivity of PLK1 at a higher EC₅₀ than for PLK2; and/or c) does notreduce activity of PLK3 in a cell expressing PLK3, or reduces activityof PLK3 at a higher EC₅₀ than for PLK2; and/or d) does not reduceactivity of PLK4 in a cell expressing PLK4, or reduces activity of PLK4at a higher EC₅₀ than for PLK2.
 23. The method of claim 22 wherein thecell is a mammalian cell overexpressing alpha-synuclein.
 24. The methodclaim 23 comprising selecting an agent that a) reduces activity of PLK2in a cell expressing PLK2; b) does not reduce activity of PLK1 in a cellexpressing PLK1, or reduces activity of PLK1 at a higher EC₅₀ than forPLK2; c) does not reduce activity of PLK3 in a cell expressing PLK3, orreduces activity of PLK3 at a higher EC₅₀ than for PLK2; and d) does notreduce activity of PLK4 in a cell expressing PLK4, or reduces activityof PLK4 at a higher EC₅₀ than for PLK2.
 25. A method of identifying anagent for treatment of Parkinson's disease comprising selecting an agentaccording to the method of claim 22, and further comprising determiningwhether the selected agent shows activity useful in treating Lewy BodyDisease in an animal model of the disease.
 26. A method of identifyingan agent for treatment of Parkinson's disease comprising selecting anagent according to the method of claim 22, and further comprisingdetermining whether the selected agent shows activity useful in treatingLewy Body Disease in a cellular model of the disease.
 27. The method ofclaim 26 wherein the cellular model of the disease is aneuronally-derived cell culture.
 28. The method of claim 27 wherein thecellular model of the disease is a mammalian cell over-expressingalpha-synuclein.
 29. The method of claim 27 wherein the activity isreduction of the proportion of total alpha-synuclein that isphosphorylated at of serine-129.
 30. The method of claim 27 wherein saidactivity is a reduction in aggregation of alpha-synuclein in the cell.31. The method of claim 22, further comprising identifying an agent thatmodulates PLK2 in the presence of synphilin.